High throughput screening of crystallization of materials

ABSTRACT

High throughput screening of crystallization of a target material is accomplished by simultaneously introducing a solution of the target material into a plurality of chambers of a microfabricated fluidic device. The microfabricated fluidic device is then manipulated to vary the solution condition in the chambers, thereby simultaneously providing a large number of crystallization environments. Control over changed solution conditions may result from a variety of techniques, including but not limited to metering volumes of crystallizing agent into the chamber by volume exclusion, by entrapment of volumes of crystallizing agent determined by the dimensions of the microfabricated structure, or by cross-channel injection of sample and crystallizing agent into an array of junctions defined by intersecting orthogonal flow channels.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of U.S. nonprovisional patentapplication Ser. No. 09/887,997 filed Jun. 22, 2001 which is acontinuation-in-part Ser. No. 09/826,583 filed Apr. 6, 2001, now U.S.Pat. No. 6,899,137, which is a continuation-in-part of Ser. No.09/724,784 filed Nov. 28, 2000 and is a continuation-in-part of Ser. No.09/605,520 filed Jun. 27, 2000. This patent application also claimspriority from U.S. provisional patent application No. 60/323,524 filedSep. 17, 2001. These prior patent applications are hereby incorporatedby reference for all purposes.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH AND DEVELOPMENT

Work described herein has been supported, in part, by National Instituteof Health grant HG-01642-02. The United States Government may thereforehave certain rights in the invention.

BACKGROUND OF THE INVENTION

Crystallization is an important technique to the biological and chemicalarts. Specifically, a high-quality crystal of a target compound can beanalyzed by x-ray diffraction techniques to produce an accuratethree-dimensional structure of the target. This three-dimensionalstructure information can then be utilized to predict functionality andbehavior of the target.

In theory, the crystallization process is simple. A target compound inpure form is dissolved in solvent. The chemical environment of thedissolved target material is then altered such that the target is lesssoluble and reverts to the solid phase in crystalline form. This changein chemical environment typically accomplished by introducing acrystallizing agent that makes the target material is less soluble,although changes in temperature and pressure can also influencesolubility of the target material.

In practice however, forming a high quality crystal is generallydifficult and sometimes impossible, requiring much trial and error andpatience on the part of the researcher. Specifically, the highly complexstructure of even simple biological compounds means that they are notamenable to forming a highly ordered crystalline structure. Therefore, aresearcher must be patient and methodical, experimenting with a largenumber of conditions for crystallization, altering parameters such assample concentration, solvent type, countersolvent type, temperature,and duration in order to obtain a high quality crystal, if in fact acrystal can be obtained at all.

Accordingly, there is a need in the art for methods and structures forperforming high throughput screening of crystallization of targetmaterials.

SUMMARY OF THE INVENTION

The present invention sets forth method and structures for performinghigh throughput screening of crystallization of target materials.Methods and structures for purifying small samples by recrystallizationare also provided.

High throughput screening of crystallization of a target material isaccomplished by simultaneously introducing a solution of the targetmaterial at a known concentration into a plurality of chambers of amicrofabricated fluidic device. The microfabricated fluidic device isthen manipulated to vary the solvent concentration in each of thechambers, thereby simultaneously providing a large number ofcrystallization environments. Control over changed solvent conditionsmay result from a variety of techniques, including but not limited tometering of a crystallizing agent through exclusion of volume from thechamber, entrapment of precisely controlled volumes of crystallizingagent as determined by the dimensions of the microfluidic device, orcross-channel injection into an array of junctions defined byintersecting orthogonal flow channels.

An embodiment of a method of metering a volume of a crystallizing agentto promote crystallization in accordance with the present inventioncomprises providing a chamber having a volume in an elastomeric blockseparated from a control recess by an elastomeric membrane, andsupplying a pressure to the control recess such that the membrane isdeflected into the chamber and the volume is reduced by a calibratedamount, thereby excluding from the chamber a calibrated volume of acrystallization sample. This method may further comprise providing asecond fluid to an opening of the chamber, and ceasing application ofthe pressure such that the membrane relaxes back to an original positionand the calibrated volume of a crystallizing agent is drawn into thechamber. This method may also further comprise the parallelization ofmultiple chambers with varying calibrated volumes.

An embodiment of a system for crystallizing a target material inaccordance with the present invention comprises an elastomeric blockincluding a microfabricated chamber configured to contain a volume of asolution of the target material, and a microfabricated flow channel influid communication with the chamber, the flow channel introducing avolume of a crystallizing agent into the chamber. The crystallizationsystem may further comprise an isolation structure configured toselectively isolate the chamber from the flow channel as the flowchannel receives a volume of a crystallizing agent, and then to placethe chamber into contact with the flow channel to alter a solutioncondition within the chamber. Alternatively, the crystallization systemmay further comprise a control channel overlying the chamber andseparated from the chamber by a membrane, the membrane deflectable intothe chamber to exclude a calibrated volume of sample solution from thechamber, such that relaxation of the membrane draws the calibratedvolume of the crystallizing agent into the chamber. Furtheralternatively, the crystallization system may comprise a plurality offirst parallel flow channels in fluid communication with a targetmaterial, and a plurality of second parallel flow channels orthogonal toand intersecting the first flow channels to create a plurality ofjunctions, the second flow channels in fluid communication with acrystallizing agent such that an array of solution environments can becreated at the junctions.

Another embodiment of a system for crystallizing a target material inaccordance with the present invention comprises an elastomeric blockincluding a microfabricated chamber configured to contain a volume of asolution of the target material, and a crystallizing agent reservoir influid communication with the microfabricated chamber through a dialysismembrane, the dialysis membrane configured to prevent flow of the targetmaterial into the crystallizing agent reservoir. The crystallizing agentreservoir may be formed in a second elastomeric block, the dialysismembrane may be present within the elastomeric block, and the dialysismembrane may comprise a polymer introduced between the chamber and thereservoir and then subjected to cross-linking.

An embodiment of a method for crystallizing a target material inaccordance with the present invention comprises charging a chamber of amicrofabricated elastomeric block with a volume of solution of thetarget material; and introducing a volume of a crystallizing agent intothe chamber to change a solvent environment of the chamber. The volumeof crystallizing agent may be introduced into the chamber by deformingan elastomer membrane overlying the chamber to exclude the volume of thesample from the chamber, followed by relaxing the membrane to cause thevolume of a surrounding crystallizing agent to flow into the chamber.Alternatively, the volume of crystallizing agent may be introduced intothe chamber by entrapping a volume of crystallizing agent proximate tothe chamber, and then opening an elastomer valve positioned between thechamber and the crystallizing agent to allow diffusion of crystallizingagent into the chamber. Further alternatively, the volume ofcrystallizing agent may be introduced into the chamber by diffusionacross a dialysis membrane.

Still further alternatively, the chamber may be defined by a junctionbetween a first flow channel orthogonal to a second flow channel, andwherein the sample is flowed through the first flow channel and thecrystallizing agent flowed through the second flow channel. An array ofsuch chambers may be defined by a junction between a first set ofparallel flow channels orthogonal to a second set of parallel flowchannels, with samples flowed through the first flow channels andcrystallizing agent flowed through the second flow channels to create anarray of solution conditions.

An embodiment of a method for crystallizing a target material comprisesintroducing a crystallizing agent to a target material solution in thepresence of a surface having a morphology calculated to serve as atemplate for formation of a crystal of the target material. In certainembodiments, this morphology may take the form of a regular morphologyof a mineral surface, or features of a semiconductor substrate patternedby lithography.

An embodiment of a method for crystallizing a target material by vapordiffusion in accordance with the present invention comprises providing atarget material solution within a microfabricated chamber, and providinga recrystallizing agent in fluid communication with the microfabricatedchamber. An air pocket is provided between the chamber and therecrystallization agent, such that the crystallizing agent diffuses inthe vapor phase across the air pocket into the target material solution.In certain embodiments, the air pocket may be secured in place throughformation of a hydrophobic material utilizing microcontact printingtechniques.

These and other embodiments of the present invention, as well as itsadvantages and features, are described in more detail in conjunctionwith the text below and attached figures.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an illustration of a first elastomeric layer formed on top ofa micromachined mold.

FIG. 2 is an illustration of a second elastomeric layer formed on top ofa micromachined mold.

FIG. 3 is an illustration of the elastomeric layer of FIG. 2 removedfrom the micromachined mold and positioned over the top of theelastomeric layer of FIG. 1

FIG. 4 is an illustration corresponding to FIG. 3, but showing thesecond elastomeric layer positioned on top of the first elastomericlayer.

FIG. 5 is an illustration corresponding to FIG. 4, but showing the firstand second elastomeric layers bonded together.

FIG. 6 is an illustration corresponding to FIG. 5, but showing the firstmicromachined mold removed and a planar substrate positioned in itsplace.

FIG. 7A is an illustration corresponding to FIG. 6, but showing theelastomeric structure sealed onto the planar substrate.

FIG. 7B is a front sectional view corresponding to FIG. 7A, showing anopen flow channel.

FIGS. 7C–7G are illustrations showing steps of a method for forming anelastomeric structure having a membrane formed from a separateelastomeric layer.

FIG. 7H is a front sectional view showing the valve of FIG. 7B in anactuated state.

FIGS. 8A and 8B illustrates valve opening vs. applied pressure forvarious flow channels.

FIG. 9 illustrates time response of a 100 μm×100 μm×10 μm RTVmicrovalve.

FIG. 10 is a front sectional view of the valve of FIG. 7B showingactuation of the membrane.

FIG. 11 is a front sectional view of an alternative embodiment of avalve having a flow channel with a curved upper surface.

FIG. 12A is a top schematic view of an on/off valve.

FIG. 12B is a sectional elevation view along line 23B—23B in FIG. 12A

FIG. 13A is a top schematic view of a peristaltic pumping system.

FIG. 13B is a sectional elevation view along line 24B—24B in FIG. 13A

FIG. 14 is a graph showing experimentally achieved pumping rates vs.frequency for an embodiment of the peristaltic pumping system of FIG.13.

FIG. 15A is a top schematic view of one control line actuating multipleflow lines simultaneously.

FIG. 15B is a sectional elevation view along line 26B—26B in FIG. 15A

FIG. 16 is a schematic illustration of a multiplexed system adapted topermit flow through various channels.

FIG. 17A is a plan view of a flow layer of an addressable reactionchamber structure.

FIG. 17B is a bottom plan view of a control channel layer of anaddressable reaction chamber structure.

FIG. 17C is an exploded perspective view of the addressable reactionchamber structure formed by bonding the control channel layer of FIG.17B to the top of the flow layer of FIG. 17A.

FIG. 17D is a sectional elevation view corresponding to FIG. 17C, takenalong line 28D—28D in FIG. 17C.

FIG. 18 is a schematic of a system adapted to selectively direct fluidflow into any of an array of reaction wells.

FIG. 19 is a schematic of a system adapted for selectable lateral flowbetween parallel flow channels.

FIG. 20A is a bottom plan view of first layer (i.e.: the flow channellayer) of elastomer of a switchable flow array.

FIG. 20B is a bottom plan view of a control channel layer of aswitchable flow array.

FIG. 20C shows the alignment of the first layer of elastomer of FIG. 20Awith one set of control channels in the second layer of elastomer ofFIG. 20B.

FIG. 20D also shows the alignment of the first layer of elastomer ofFIG. 20A with the other set of control channels in the second layer ofelastomer of FIG. 20B.

FIGS. 21A–21J show views of one embodiment of a normally-closed valvestructure in accordance with the present invention.

FIGS. 22A and 22B show plan views illustrating operation of oneembodiment of a side-actuated valve structure in accordance with thepresent invention.

FIG. 23 shows a cross-sectional view of one embodiment of a compositestructure in accordance with the present invention.

FIG. 24 shows a cross-sectional view of another embodiment of acomposite structure in accordance with the present invention.

FIG. 25 shows a cross-sectional view of another embodiment of acomposite structure in accordance with the present invention.

FIGS. 26A–26D show plan views illustrating operation of one embodimentof a cell pen structure in accordance with the present invention.

FIGS. 27A–27B show plan and cross-sectional views illustrating operationof one embodiment of a cell cage structure in accordance with thepresent invention.

FIGS. 28A–28B show plan views of operation of a wiring structureutilizing cross-channel injection in accordance with the embodiment ofthe present invention.

FIGS. 29A–29D illustrate cross-sectional views of metering by volumeexclusion in accordance with an embodiment of the present invention.

FIG. 30 is a plan view of one embodiment of a recrystallization systemin accordance with one embodiment of the present invention utilizingvolume exclusion.

FIG. 31 is a plan view of one embodiment of a recrystallization systemin accordance with the present invention utilizing volume entrapment.

FIG. 32 is a plan view of an alternative embodiment of arecrystallization system in accordance with the present inventionutilizing volume entrapment.

FIG. 33 is a plan view of a protein crystallization system in allowancewith one embodiment in accordance with the present invention utilizingcross-channel injection.

FIGS. 34A–34C are enlarged views of a portion of the recrystallizationsystem of FIG. 32 showing its operation.

FIG. 35 is a cross-sectional view of one embodiment of arecrystallization system in accordance with the present inventionutilizing a dialysis membrane.

FIG. 36 is a cross-sectional view of another embodiment of arecrystallization system in accordance with the present inventionutilizing a dialysis membrane.

FIG. 37 is a plan view of still another embodiment of arecrystallization system in accordance with the present inventionutilizing a dialysis membrane.

FIGS. 38A–C show cross-sectional views of a process for formingelastomer structures by bonding along a vertical line.

FIG. 39 shows a plan view of an embodiment of a structure in accordancewith the present invention for performing crystallization by vapor phasediffusion.

FIG. 40 shows a plan view of another embodiment of a structure inaccordance with the present invention for performing crystallization byvapor phase diffusion.

FIG. 41 shows a plan view of still another embodiment of a structure inaccordance with the present invention for performing crystallization byvapor phase diffusion.

FIG. 42 shows a plan view of an embodiment of a structure in accordancewith the present invention for sorting crystals of various sizes.

FIG. 43 is a plan view of an alternative embodiment of arecrystallization system in accordance with the present inventionutilizing volume entrapment.

FIG. 44 plots Log(R/B) vs. number of slugs injected for one embodimentof a cross-flow injection system in accordance with the presentinvention.

FIG. 45A shows a simplified plan view of the alternative embodiment ofthe chip utilized to obtain experimental results.

FIG. 45B shows as simplified enlarged plan view of a set of threecompound wells of the chip shown in FIG. 45A.

FIG. 45C shows a simplified cross-sectional view of the compound wellsof FIGS. 45A–B.

FIG. 46 shows a Venn diagram summarizing the results of one set ofexperiments for crystallization of glucose isomerase.

FIG. 47A shows a photograph of a salt crystal. FIG. 47B shows photographof a crumbled protein crystal.

FIGS. 48A–B show photographs of large, high quality glucose isomerasecrystals formed a chip.

FIG. 49 shows a Venn diagram summarizing a second set of experiments forcrystallization of glucose isomerase.

FIG. 50 shows photographs of a number of glucose isomerase crystalsformed utilizing the Hampton condition IS.

FIG. 51 shows additional photographs of glucose isomerase crystals.

FIG. 52 is a Venn diagram summarizing experiments for crystallization ofproteinase K.

FIG. 53A shows a proteinase K crystal formed by conventional microbatch.

FIG. 53B shows proteinase K crystals observed on the chip.

FIG. 54 shows photographs of additional proteinase K crystals.

FIG. 55 is a Venn diagram summarizing experiments for crystallization ofthe beef liver catalase protein.

FIG. 56A shows a photograph of the beef liver catalase protein crystalsformed by conventional microbatch techniques. FIG. 56B shows aphotograph of beef liver catalase protein crystals formed on a chip inaccordance with an embodiment of the present invention.

FIG. 57 is a Venn diagram summarizing experiments for crystallization ofthe bovine pancreas trypsin protein.

FIG. 58A shows a photograph of bovine pancreas trypsin crystals producedby microbatch. FIG. 58B shows bovine pancreas trypsin crystals formed ona chip in accordance with one embodiment of the present invention.

FIG. 59 is a Venn diagram summarizing experiments for crystallization oflysozyme.

FIG. 60A shows a photograph of a lysozyme crystal formed on a chip inaccordance with one embodiment of the present invention. FIG. 60B showsa photograph of a lysozyme crystal formed utilizing a conventionalmicrobatch method.

FIG. 61 is a Venn diagram summarizing experiments for crystallization ofXylanase.

FIG. 62A shows a photograph of a Xylanase crystal formed utilizing aconventional microbatch technique. FIG. 62B shows a photograph of aXylanase crystal formed on a chip in accordance with an embodiment ofthe present invention.

FIGS. 63A–B show photographs of Xylanase crystals formed utilizing achip in accordance with one embodiment of the present invention.

FIGS. 64A–B show photographs of large, high quality crystals of the Bsubunit of topoisomerase VI protein formed utilizing a ship inaccordance with one embodiment of the present invention.

FIG. 65 plots a bar graph of crystallization hits generated on thevarious proteins for the conventional microbatch and hanging droptechniques, and for a chip in accordance with one embodiment of thepresent invention.

FIG. 66 compares the phase space evolution and equilibration of theconventional microbatch and hanging drop methods, and for themicrofluidic device in accordance with the embodiment of the presentinvention.

FIGS. 67A–C show photographs of equilibrium of dyes present in threecompared wells of the chip shown in FIG. 45, at elapsed diffusion timesof 0s, 10 min, and 45 min.

FIG. 68A shows a photograph of crystal formation in silicone oil. FIG.68B shows a photograph evidencing the absence of crystal formation inparaffin oil.

FIG. 69 shows a photograph of the Xylanase crystal of FIG. 62B grownon-chip, as mounted in a cryo-loop.

FIG. 70A shows a plan view of one embodiment of a crystallizationgrowing chip in accordance with an embodiment of the present invention.

FIG. 70B shows a simplified cross-sectional view of the embodiment ofthe crystal growing chip shown in FIG. 70A along line B—B.

FIGS. 71A–D are simplified schematic drawings plotting concentrationversus distance from a free interface.

FIGS. 72A–B show simplified cross-sectional views of the attemptedformation of a microscopic free interface in a capillary tube.

FIGS. 73A–B show mixing of solutions in a capillary tube as a result ofthe parabolic velocity distribution of pressure driven Poiseuille flow.

FIGS. 74A–C show the microscopic free interface formed by solutionshaving different densities.

FIGS. 75A–D show a simplified schematic view of the formation of a highquality microfluidic free interface resulting from pressurized out-gaspriming (POP) of a microfluidic structure in accordance with anembodiment of the present invention.

FIG. 76A shows a simplified plan view of two microfluidic chamber whosecommunication through a flow channel is controlled by a valve.

FIG. 76B plots concentration of the first solution in the first chamberversus time flowing actuation of the interface valve. FIG. 76C plotsconcentration of the first solution in the first chamber versus timefollowing actuation of the interface valve at 50% the duty cycle of FIG.76B.

FIG. 77A shows three sets of pairs of compound chambers connected bymicrochannels of different length. FIG. 77B plots equilibration timeversus equilibration distance.

FIG. 78 shows four compound chambers with different connecting microchannels.

FIG. 79A shows a microfluidic architecture designed to capture aconcentration gradient over a distance. FIG. 79B plots concentrationversus distance from a free interface at an initial time. FIG. 79C plotsconcentration versus distance from a free interface at a subsequenttime. FIG. 79D plots relative chamber concentration versus distance atthe subsequent time.

FIG. 80 shows an exploded view of one embodiment of a chip holder devicein accordance with the present invention.

FIG. 81A shows an enlarged view of a portion of one flow channel inaccordance with an alternative embodiment of the present invention. FIG.81B shows cross-sectional view along line B–B′ of the enlarged flowchannel portion of FIG. 81A prior to deactuation of alternative rowvalves to allow diffusion between crystallizing agent and targetmaterial in adjacent chambers. FIG. 81C shows cross-sectional view alongline B–B′ of the enlarged flow channel portion of FIG. 81A afterdeactuation of alternative row valves to allow diffusion betweencrystallizing agent and target material in adjacent chambers.

FIG. 82 plots injected volume versus number of injection cycles forcross-channel flow injection over a variety of flow conditions.

DETAILED DESCRIPTION OF THE PRESENT INVENTION

I. Microfabrication Overview

The following discussion relates to formation of microfabricated fluidicdevices utilizing elastomer materials, as described generally in U.S.patent application Ser. No. 09/826,585 filed Apr. 6, 2001, Ser. No.09/724,784 filed Nov. 28, 2000, and Ser. No. 09/605,520, filed Jun. 27,2000. These patent applications are hereby incorporated by reference.

1. Methods of Fabricating

Exemplary methods of fabricating the present invention are providedherein. It is to be understood that the present invention is not limitedto fabrication by one or the other of these methods. Rather, othersuitable methods of fabricating the present microstructures, includingmodifying the present methods, are also contemplated.

FIGS. 1 to 7B illustrate sequential steps of a first preferred method offabricating the present microstructure, (which may be used as a pump orvalve). FIGS. 8 to 18 illustrate sequential steps of a second preferredmethod of fabricating the present microstructure, (which also may beused as a pump or valve).

As will be explained, the preferred method of FIGS. 1 to 7B involvesusing pre-cured elastomer layers which are assembled and bonded. In analternative method, each layer of elastomer may be cured “in place”. Inthe following description “channel” refers to a recess in theelastomeric structure which can contain a flow of fluid or gas.

Referring to FIG. 1, a first micro-machined mold 10 is provided.Micro-machined mold 10 may be fabricated by a number of conventionalsilicon processing methods, including but not limited tophotolithography, ion-milling, and electron beam lithography.

As can be seen, micro-machined mold 10 has a raised line or protrusion11 extending therealong. A first elastomeric layer 20 is cast on top ofmold 10 such that a first recess 21 will be formed in the bottom surfaceof elastomeric layer 20, (recess 21 corresponding in dimension toprotrusion 11), as shown.

As can be seen in FIG. 2, a second micro-machined mold 12 having araised protrusion 13 extending therealong is also provided. A secondelastomeric layer 22 is cast on top of mold 12, as shown, such that arecess 23 will be formed in its bottom surface corresponding to thedimensions of protrusion 13.

As can be seen in the sequential steps illustrated in FIGS. 3 and 4,second clastomeric layer 22 is then removed from mold 12 and placed ontop of first elastomeric layer 20. As can be seen, recess 23 extendingalong the bottom surface of second elastomeric layer 22 will form a flowchannel 32.

Referring to FIG. 5, the separate first and second elastomeric layers 20and 22 (FIG. 4) are then bonded together to form an integrated (i.e.:monolithic) elastomeric structure 24.

As can been seen in the sequential step of FIGS. 6 and 7A, elastomericstructure 24 is then removed from mold 10 and positioned on top of aplanar substrate 14.

As can be seen in FIGS. 7A and 7B, when elastomeric structure 24 hasbeen sealed at its bottom surface to planar substrate 14, recess 21 willform a flow channel 30.

The present elastomeric structures form a reversible hermetic seal withnearly any smooth planar substrate. An advantage to forming a seal thisway is that the elastomeric structures may be peeled up, washed, andre-used. In preferred aspects, planar substrate 14 is glass. A furtheradvantage of using glass is that glass is transparent, allowing opticalinterrogation of elastomer channels and reservoirs. Alternatively, theelastomeric structure may be bonded onto a flat elastomer layer by thesame method as described above, forming a permanent and high-strengthbond. This may prove advantageous when higher back pressures are used.

As can be seen in FIG. 7A and 7B, flow channels 30 and 32 are preferablydisposed at an angle to one another with a small membrane 25 ofsubstrate 24 separating the top of flow channel 30 from the bottom offlow channel 32.

In preferred aspects, planar substrate 14 is glass. An advantage ofusing glass is that the present elastomeric structures may be peeled up,washed and reused. A further advantage of using glass is that opticalsensing may be employed. Alternatively, planar substrate 14 may be anelastomer itself, which may prove advantageous when higher backpressures are used.

The method of fabrication just described may be varied to form astructure having a membrane composed of an elastomeric materialdifferent than that forming the walls of the channels of the device.This variant fabrication method is illustrated in FIGS. 7C–7G.

Referring to FIG. 7C, a first micro-machined mold 10 is provided.Micro-machined mold 10 has a raised line or protrusion 11 extendingtherealong. In FIG. 7D, first elastomeric layer 20 is cast on top offirst micro-machined mold 10 such that the top of the first elastomericlayer 20 is flush with the top of raised line or protrusion 11. This maybe accomplished by carefully controlling the volume of elastomericmaterial spun onto mold 10 relative to the known height of raised line11. Alternatively, the desired shape could be formed by injectionmolding.

In FIG. 7E, second micro-machined mold 12 having a raised protrusion 13extending therealong is also provided. Second elastomeric layer 22 iscast on top of second mold 12 as shown, such that recess 23 is formed inits bottom surface corresponding to the dimensions of protrusion 13.

In FIG. 7F, second elastomeric layer 22 is removed from mold 12 andplaced on top of third elastomeric layer 222. Second elastomeric layer22 is bonded to third elastomeric layer 20 to form integral elastomericblock 224 using techniques described in detail below. At this point inthe process, recess 23 formerly occupied by raised line 13 will formflow channel 23.

In FIG. 7G, elastomeric block 224 is placed on top of firstmicro-machined mold 10 and first elastomeric layer 20. Elastomeric blockand first elastomeric layer 20 are then bonded together to form anintegrated (i.e.: monolithic) elastomeric structure 24 having a membranecomposed of a separate elastomeric layer 222.

When elastomeric structure 24 has been sealed at its bottom surface to aplanar substrate in the manner described above in connection with FIG.7A, the recess formerly occupied by raised line 11 will form flowchannel 30.

The variant fabrication method illustrated above in conjunction withFIGS. 7C–7G offers the advantage of permitting the membrane portion tobe composed of a separate material than the elastomeric material of theremainder of the structure. This is important because the thickness andelastic properties of the membrane play a key role in operation of thedevice. Moreover, this method allows the separate elastomer layer toreadily be subjected to conditioning prior to incorporation into theelastomer structure. As discussed in detail below, examples ofpotentially desirable condition include the introduction of magnetic orelectrically conducting species to permit actuation of the membrane,and/or the introduction of dopant into the membrane in order to alterits elasticity.

While the above method is illustrated in connection with forming variousshaped elastomeric layers formed by replication molding on top of amicromachined mold, the present invention is not limited to thistechnique. Other techniques could be employed to form the individuallayers of shaped elastomeric material that are to be bonded together.For example, a shaped layer of elastomeric material could be formed bylaser cutting or injection molding, or by methods utilizing chemicaletching and/or sacrificial materials as discussed below in conjunctionwith the second exemplary method.

An alternative method fabricates a patterned elastomer structureutilizing development of photoresist encapsulated within elastomermaterial. However, the methods in accordance with the present inventionare not limited to utilizing photoresist. Other materials such as metalscould also serve as sacrificial materials to be removed selective to thesurrounding elastomer material, and the method would remain within thescope of the present invention. For example, gold metal may be etchedselective to RTV 615 elastomer utilizing the appropriate chemicalmixture.

2. Layer and Channel Dimensions

Microfabricated refers to the size of features of an elastomericstructure fabricated in accordance with an embodiment of the presentinvention. In general, variation in at least one dimension ofmicrofabricated structures is controlled to the micron level, with atleast one dimension being microscopic (i.e. below 1000 μm).Microfabrication typically involves semiconductor or MEMS fabricationtechniques such as photolithography and spincoating that are designedfor to produce feature dimensions on the microscopic level, with atleast some of the dimension of the microfabricated structure requiring amicroscope to reasonably resolve/image the structure.

In preferred aspects, flow channels 30, 32, 60 and 62 preferably havewidth-to-depth ratios of about 10:1. A non-exclusive list of otherranges of width-to-depth ratios in accordance with embodiments of thepresent invention is 0.1:1 to 100:1, more preferably 1:1 to 50:1, morepreferably 2:1 to 20:1, and most preferably 3:1 to 15:1. In an exemplaryaspect, flow channels 30, 32, 60 and 62 have widths of about 1 to 1000microns. A non-exclusive list of other ranges of widths of flow channelsin accordance with embodiments of the present invention is 0.01 to 1000microns, more preferably 0.05 to 1000 microns, more preferably 0.2 to500 microns, more preferably 1 to 250 microns, and most preferably 10 to200 microns. Exemplary channel widths include 0.1 μm, 1 μm, 2 μm, 5 μm,10 μm, 20 μm, 30 μm, 40 μm, 50 μm, 60 μm, 70 μm, 80 μm, 90 μm, 100 μm,110 μm, 120 μm, 130 μm, 140 μm, 150 μm, 160 μm, 170 μm, 180 μm, 190 μm,200 μm, 210 μm, 220 μm, 230 μm, 240 μm, and 250 μm.

Flow channels 30, 32, 60, and 62 have depths of about 1 to 100 microns.A non-exclusive list of other ranges of depths of flow channels inaccordance with embodiments of the present invention is 0.01 to 1000microns, more preferably 0.05 to 500 microns, more preferably 0.2 to 250microns, and more preferably 1 to 100 microns, more preferably 2 to 20microns, and most preferably 5 to 10 microns. Exemplary channel depthsinclude including 0.01 μm, 0.02 μm, 0.05 μm, 0.1 μm, 0.2 μm, 0.5 82 m, 1μm, 2 μm, 3 μm, 4 μm, 5 μm, 7.5 μm, 10 μm, 12.5 μm, 15 μm, 17.5 μm, 20μm, 22.5 μm, 25 μm, 30 μm, 40 μm, 50 μm, 75 μm, 100 μm, 150 μm, 200 μm,and 250 μm

The flow channels are not limited to these specific dimension ranges andexamples given above, and may vary in width in order to affect themagnitude of force required to deflect the membrane as discussed atlength below in conjunction with FIG. 27. For example, extremely narrowflow channels having a width on the order of 0.01 μm may be useful inoptical and other applications, as discussed in detail below.Elastomeric structures which include portions having channels of evengreater width than described above are also contemplated by the presentinvention, and examples of applications of utilizing such wider flowchannels include fluid reservoir and mixing channel structures.

The Elastomeric layers may be cast thick for mechanical stability. In anexemplary embodiment, elastomeric layer 22 of FIG. 1 is 50 microns toseveral centimeters thick, and more preferably approximately 4 mm thick.A non-exclusive list of ranges of thickness of the elastomer layer inaccordance with other embodiments of the present invention is betweenabout 0.1 micron to 10 cm, 1 micron to 5 cm, 10 microns to 2 cm, 100microns to 10 mm.

Accordingly, membrane 25 of FIG. 7B separating flow channels 30 and 32has a typical thickness of between about 0.01 and 1000 microns, morepreferably 0.05 to 500 microns, more preferably 0.2 to 250, morepreferably 1 to 100 microns, more preferably 2 to 50 microns, and mostpreferably 5 to 40 microns. As such, the thickness of clastomeric layer22 is about 100 times the thickness of elastomeric layer 20. Exemplarymembrane thicknesses include 0.01 μm, 0.02 μm, 0.03 μm, 0.05 μm, 0.1 μm,0.2 μm, 0.3 μm, 0.5 μm, 1 μm, 2 μm, 3 μm, 5 μm, 7.5 μm, 10 μm, 12.5 μm,15 μm, 17.5 μm, 20 μm, 22.5 μm, 25 μm, 30 μm, 40 μm, 50 μm, 75 μm, 100μm, 150 μm, 200 μm, 250 μm, 300 μm, 400 μm, 500 μm, 750 μm, and 1000 μm.

3. Soft Lithographic Bonding

Preferably, elastomeric layers are bonded together chemically, usingchemistry that is intrinsic to the polymers comprising the patternedelastomer layers. Most preferably, the bonding comprises two component“addition cure” bonding.

In a preferred aspect, the various layers of clastomer are boundtogether in a heterogenous bonding in which the layers have a differentchemistry. Alternatively, a homogenous bonding may be used in which alllayers would be of the same chemistry. Thirdly, the respective elastomerlayers may optionally be glued together by an adhesive instead. In afourth aspect, the elastomeric layers may be thermoset elastomers bondedtogether by heating.

In one aspect of homogeneous bonding, the elastomeric layers arecomposed of the same elastomer material, with the same chemical entityin one layer reacting with the same chemical entity in the other layerto bond the layers together. In one embodiment, bonding between polymerchains of like elastomer layers may result from activation of acrosslinking agent due to light, heat, or chemical reaction with aseparate chemical species.

Alternatively in a heterogeneous aspect, the elastomeric layers arecomposed of different elastomeric materials, with a first chemicalentity in one layer reacting with a second chemical entity in anotherlayer. In one exemplary heterogenous aspect, the bonding process used tobind respective elastomeric layers together may comprise bondingtogether two layers of RTV 615 silicone. RTV 615 silicone is a two-partaddition-cure silicone rubber. Part A contains vinyl groups andcatalyst; part B contains silicon hydride (Si—H) groups. Theconventional ratio for RTV 615 is 10A:1B. For bonding, one layer may bemade with 30A:1B (i.e. excess vinyl groups) and the other with 3A:1B(i.e. excess Si—H groups). Each layer is cured separately. When the twolayers are brought into contact and heated at elevated temperature, theybond irreversibly forming a monolithic elastomeric substrate.

In an exemplary aspect of the present invention, elastomeric structuresare formed utilizing Sylgard 182, 184 or 186, or aliphatic urethanediacrylates such as (but not limited to) Ebecryl 270 or Irr 245 from UCBChemical.

In one embodiment in accordance with the present invention, two-layerelastomeric structures were fabricated from pure acrylated Urethane Ebe270. A thin bottom layer was spin coated at 8000 rpm for 15 seconds at170° C. The top and bottom layers were initially cured under ultravioletlight for 10 minutes under nitrogen utilizing a Model ELC 500 devicemanufactured by Electrolite corporation. The assembled layers were thencured for an additional 30 minutes. Reaction was catalyzed by a 0.5%vol/vol mixture of Irgacure 500 manufactured by Ciba-Geigy Chemicals.The resulting elastomeric material exhibited moderate elasticity andadhesion to glass.

In another embodiment in accordance with the present invention,two-layer elastomeric structures were fabricated from a combination of25% Ebe 270/50% Irr245/25% isopropyl alcohol for a thin bottom layer,and pure acrylated Urethane Ebe 270 as a top layer. The thin bottomlayer was initially cured for 5 min, and the top layer initially curedfor 10 minutes, under ultraviolet light under nitrogen utilizing a ModelELC 500 device manufactured by Electrolite corporation. The assembledlayers were then cured for an additional 30 minutes. Reaction wascatalyzed by a 0.5% vol/vol mixture of Irgacure 500 manufactured byCiba-Geigy Chemicals. The resulting elastomeric material exhibitedmoderate elasticity and adhered to glass.

Alternatively, other bonding methods may be used, including activatingthe elastomer surface, for example by plasma exposure, so that theelastomer layers/substrate will bond when placed in contact. Forexample, one possible approach to bonding together elastomer layerscomposed of the same material is set forth by Duffy et al, “RapidPrototyping of Microfluidic Systems in Poly (dimethylsiloxane)”,Analytical Chemistry (1998), 70, 4974–4984, incorporated herein byreference. This paper discusses that exposing polydimethylsiloxane(PDMS) layers to oxygen plasma causes oxidation of the surface, withirreversible bonding occurring when the two oxidized layers are placedinto contact.

Yet another approach to bonding together successive layers of elastomeris to utilize the adhesive properties of uncured elastomer.Specifically, a thin layer of uncured elastomer such as RTV 615 isapplied on top of a first cured elastomeric layer. Next, a second curedelastomeric layer is placed on top of the uncured elastomeric layer. Thethin middle layer of uncured elastomer is then cured to produce amonolithic elastomeric structure. Alternatively, uncured elastomer canbe applied to the bottom of a first cured clastomer layer, with thefirst cured elastomer layer placed on top of a second cured elastomerlayer. Curing the middle thin elastomer layer again results in formationof a monolithic elastomeric structure.

Where encapsulation of sacrificial layers is employed to fabricate theelastomer structure, bonding of successive elastomeric layers may beaccomplished by pouring uncured elastomer over a previously curedelastomeric layer and any sacrificial material patterned thereupon.Bonding between elastomer layers occurs due to interpenetration andreaction of the polymer chains of an uncured elastomer layer with thepolymer chains of a cured elastomer layer. Subsequent curing of theelastomeric layer will create a bond between the elastomeric layers andcreate a monolithic elastomeric structure.

Referring to the first method of FIGS. 1 to 7B, first elastomeric layer20 may be created by spin-coating an RTV mixture on microfabricated mold12 at 2000 rpm's for 30 seconds yielding a thickness of approximately 40microns. Second elastomeric layer 22 may be created by spin-coating anRTV mixture on microfabricated mold 11. Both layers 20 and 22 may beseparately baked or cured at about 80° C. for 1.5 hours. The secondelastomeric layer 22 may be bonded onto first elastomeric layer 20 atabout 80° C. for about 1.5 hours.

Micromachined molds 10 and 12 may be patterned photoresist on siliconwafers. In an exemplary aspect, a Shipley SJR 5740 photoresist was spunat 2000 rpm patterned with a high resolution transparency film as a maskand then developed yielding an inverse channel of approximately 10microns in height. When baked at approximately 200° C. for about 30minutes, the photoresist reflows and the inverse channels becomerounded. In preferred aspects, the molds may be treated withtrimethylchlorosilane (TMCS) vapor for about a minute before each use inorder to prevent adhesion of silicone rubber.

4. Suitable Elastomeric Materials

Allcock et al, Contemporary Polymer Chemistry, 2^(nd) Ed. describeselastomers in general as polymers existing at a temperature betweentheir glass transition temperature and liquefaction temperature.Elastomeric materials exhibit elastic properties because the polymerchains readily undergo torsional motion to permit uncoiling of thebackbone chains in response to a force, with the backbone chainsrecoiling to assume the prior shape in the absence of the force. Ingeneral, elastomers deform when force is applied, but then return totheir original shape when the force is removed. The elasticity exhibitedby elastomeric materials may be characterized by a Young's modulus.Elastomeric materials having a Young's modulus of between about 1 Pa–1TPa, more preferably between about 10 Pa–100 GPa, more preferablybetween about 20 Pa–1 GPa, more preferably between about 50 Pa–10 MPa,and more preferably between about 100 Pa–1 MPa are useful in accordancewith the present invention, although elastomeric materials having aYoung's modulus outside of these ranges could also be utilized dependingupon the needs of a particular application.

The systems of the present invention may be fabricated from a widevariety of elastomers. In an exemplary aspect, the elastomeric layersmay preferably be fabricated from silicone rubber. However, othersuitable elastomers may also be used.

In an exemplary aspect of the present invention, the present systems arefabricated from an elastomeric polymer such as GE RTV 615 (formulation),a vinyl-silane crosslinked (type) silicone elastomer (family). However,the present systems are not limited to this one formulation, type oreven this family of polymer; rather, nearly any elastomeric polymer issuitable. An important requirement for the preferred method offabrication of the present microvalves is the ability to bond multiplelayers of elastomers together. In the case of multilayer softlithography, layers of elastomer are cured separately and then bondedtogether. This scheme requires that cured layers possess sufficientreactivity to bond together. Either the layers may be of the same type,and are capable of bonding to themselves, or they may be of twodifferent types, and are capable of bonding to each other. Otherpossibilities include the use an adhesive between layers and the use ofthermoset elastomers.

Given the tremendous diversity of polymer chemistries, precursors,synthetic methods, reaction conditions, and potential additives, thereare a huge number of possible elastomer systems that could be used tomake monolithic elastomeric microvalves and pumps. Variations in thematerials used will most likely be driven by the need for particularmaterial properties, i.e. solvent resistance, stiffness, gaspermeability, or temperature stability.

There are many, many types of elastomeric polymers. A brief descriptionof the most common classes of elastomers is presented here, with theintent of showing that even with relatively “standard” polymers, manypossibilities for bonding exist. Common elastomeric polymers includepolyisoprene, polybutadiene, polychloroprene, polyisobutylene,poly(styrene-butadiene-styrene), the polyurethanes, and silicones.

Polyisoprene, polybutadiene, polychloroprene:

Polyisoprene, polybutadiene, and polychloroprene are all polymerizedfrom diene monomers, and therefore have one double bond per monomer whenpolymerized. This double bond allows the polymers to be converted toelastomers by vulcanization (essentially, sulfur is used to formcrosslinks between the double bonds by heating). This would easily allowhomogeneous multilayer soft lithography by incomplete vulcanization ofthe layers to be bonded; photoresist encapsulation would be possible bya similar mechanism.

Polyisobutylene:

Pure Polyisobutylene has no double bonds, but is crosslinked to use asan elastomer by including a small amount (˜1%) of isoprene in thepolymerization. The isoprene monomers give pendant double bonds on thePolyisobutylene backbone, which may then be vulcanized as above.

Poly(styrene-butadiene-styrene):

Poly(styrene-butadiene-styrene) is produced by living anionicpolymerization (that is, there is no natural chain-terminating step inthe reaction), so “live” polymer ends can exist in the cured polymer.This makes it a natural candidate for the present photoresistencapsulation system (where there will be plenty of unreacted monomer inthe liquid layer poured on top of the cured layer). Incomplete curingwould allow homogeneous multilayer soft lithography (A to A bonding).The chemistry also facilitates making one layer with extra butadiene(“A”) and coupling agent and the other layer (“B”) with a butadienedeficit (for heterogeneous multilayer soft lithography). SBS is a“thermoset elastomer”, meaning that above a certain temperature it meltsand becomes plastic (as opposed to elastic); reducing the temperatureyields the elastomer again. Thus, layers can be bonded together byheating.

Polyurethanes:

Polyurethanes are produced from di-isocyanates (A—A) and di-alcohols ordi-amines (B—B); since there are a large variety of di-isocyanates anddi-alcohols/amines, the number of different types of polyurethanes ishuge. The A vs. B nature of the polymers, however, would make themuseful for heterogeneous multilayer soft lithography just as RTV 615 is:by using excess A—A in one layer and excess B—B in the other layer.

Silicones:

Silicone polymers probably have the greatest structural variety, andalmost certainly have the greatest number of commercially availableformulations. The vinyl-to-(Si—H) crosslinking of RTV 615 (which allowsboth heterogeneous multilayer soft lithography and photoresistencapsulation) has already been discussed, but this is only one ofseveral crosslinking methods used in silicone polymer chemistry.

5. Operation of Device

FIGS. 7B and 7H together show the closing of a first flow channel bypressurizing a second flow channel, with FIG. 7B (a front sectional viewcutting through flow channel 32 in corresponding FIG. 7A), showing anopen first flow channel 30; with FIG. 7H showing first flow channel 30closed by pressurization of the second flow channel 32.

Referring to FIG. 7B, first flow channel 30 and second flow channel 32are shown. Membrane 25 separates the flow channels, forming the top offirst flow channel 30 and the bottom of second flow channel 32. As canbe seen, flow channel 30 is “open”.

As can be seen in FIG. 7H, pressurization of flow channel 32 (either bygas or liquid introduced therein) causes membrane 25 to deflectdownward, thereby pinching off flow F passing through flow channel 30.Accordingly, by varying the pressure in channel 32, a linearly actuablevalving system is provided such that flow channel 30 can be opened orclosed by moving membrane 25 as desired. (For illustration purposesonly, channel 30 in FIG. 7G is shown in a “mostly closed” position,rather than a “fully closed” position).

Since such valves are actuated by moving the roof of the channelsthemselves (i.e.: moving membrane 25) valves and pumps produced by thistechnique have a truly zero dead volume, and switching valves made bythis technique have a dead volume approximately equal to the activevolume of the valve, for example about 100×100×10 μm=100 pL. Such deadvolumes and areas consumed by the moving membrane are approximately twoorders of magnitude smaller than known conventional microvalves. Smallerand larger valves and switching valves are contemplated in the presentinvention, and a non-exclusive list of ranges of dead volume includes 1aL to 1 uL, 100 aL to 100 nL, 1 fL to 10 nL, 100 fL to 1 nL, and 1 pL to100 pL.

The extremely small volumes capable of being delivered by pumps andvalves in accordance with the present invention represent a substantialadvantage. Specifically, the smallest known volumes of fluid capable ofbeing manually metered is around 0.1 μl. The smallest known volumescapable of being metered by automated systems is about ten-times larger(1 μl). Utilizing pumps and valves in accordance with the presentinvention, volumes of liquid of 10 nl or smaller can routinely bemetered and dispensed. The accurate metering of extremely small volumesof fluid enabled by the present invention would be extremely valuable ina large number of biological applications, including diagnostic testsand assays.

Equation 1 represents a highly simplified mathematical model ofdeflection of a rectangular, linear, elastic, isotropic plate of uniformthickness by an applied pressure:w=(BPb ⁴)/(Eh ³),  (1)where:

-   -   w=deflection of plate;    -   B=shape coefficient (dependent upon length vs. width and support        of edges of plate);    -   P=applied pressure;    -   b=plate width    -   E=Young's modulus; and    -   h=plate thickness.

Thus even in this extremely simplified expression, deflection of anelastomeric membrane in response to a pressure will be a function of:the length, width, and thickness of the membrane, the flexibility of themembrane (Young's modulus), and the applied actuation force. Becauseeach of these parameters will vary widely depending upon the actualdimensions and physical composition of a particular elastomeric devicein accordance with the present invention, a wide range of membranethicknesses and elasticity's, channel widths, and actuation forces arecontemplated by the present invention.

It should be understood that the formula just presented is only anapproximation, since in general the membrane does not have uniformthickness, the membrane thickness is not necessarily small compared tothe length and width, and the deflection is not necessarily smallcompared to length, width, or thickness of the membrane. Nevertheless,the equation serves as a useful guide for adjusting variable parametersto achieve a desired response of deflection versus applied force.

FIGS. 8A and 8B illustrate valve opening vs. applied pressure for a 100μm wide first flow channel 30 and a 50 μm wide second flow channel 32.The membrane of this device was formed by a layer of General ElectricSilicones RTV 615 having a thickness of approximately 30 μm and aYoung's modulus of approximately 750 kPa. FIGS. 21 a and 21 b show theextent of opening of the valve to be substantially linear over most ofthe range of applied pressures.

Air pressure was applied to actuate the membrane of the device through a10 cm long piece of plastic tubing having an outer diameter of 0.025″connected to a 25 mm piece of stainless steel hypodermic tubing with anouter diameter of 0.025″ and an inner diameter of 0.013″. This tubingwas placed into contact with the control channel by insertion into theelastomeric block in a direction normal to the control channel. Airpressure was applied to the hypodermic tubing from an external LHDAminiature solenoid valve manufactured by Lee Co.

While control of the flow of material through the device has so far beendescribed utilizing applied gas pressure, other fluids could be used.

For example, air is compressible, and thus experiences some finite delaybetween the time of application of pressure by the external solenoidvalve and the time that this pressure is experienced by the membrane. Inan alternative embodiment of the present invention, pressure could beapplied from an external source to a noncompressible fluid such as wateror hydraulic oils, resulting in a near-instantaneous transfer of appliedpressure to the membrane. However, if the displaced volume of the valveis large or the control channel is narrow, higher viscosity of a controlfluid may contribute to delay in actuation. The optimal medium fortransferring pressure will therefore depend upon the particularapplication and device configuration, and both gaseous and liquid mediaare contemplated by the invention.

While external applied pressure as described above has been applied by apump/tank system through a pressure regulator and external miniaturevalve, other methods of applying external pressure are also contemplatedin the present invention, including gas tanks, compressors, pistonsystems, and columns of liquid. Also contemplated is the use ofnaturally occurring pressure sources such as may be found inside livingorganisms, such as blood pressure, gastric pressure, the pressurepresent in the cerebro-spinal fluid, pressure present in theintra-ocular space, and the pressure exerted by muscles during normalflexure. Other methods of regulating external pressure are alsocontemplated, such as miniature valves, pumps, macroscopic peristalticpumps, pinch valves, and other types of fluid regulating equipment suchas is known in the art.

As can be seen, the response of valves in accordance with embodiments ofthe present invention have been experimentally shown to be almostperfectly linear over a large portion of its range of travel, withminimal hysteresis. Accordingly, the present valves are ideally suitedfor microfluidic metering and fluid control. The linearity of the valveresponse demonstrates that the individual valves are well modeled asHooke's Law springs. Furthermore, high pressures in the flow channel(i.e.: back pressure) can be countered simply by increasing theactuation pressure. Experimentally, the present inventors have achievedvalve closure at back pressures of 70 kPa, but higher pressures are alsocontemplated. The following is a nonexclusive list of pressure rangesencompassed by the present invention: 10 Pa–25 MPa; 100 Pa–10 Mpa, 1kPa–1 MPa, 1 kPa–300 kPa, 5 kPa–200 kPa, and 15 kPa–100 kPa.

While valves and pumps do not require linear actuation to open andclose, linear response does allow valves to more easily be used asmetering devices. In one embodiment of the invention, the opening of thevalve is used to control flow rate by being partially actuated to aknown degree of closure. Linear valve actuation makes it easier todetermine the amount of actuation force required to close the valve to adesired degree of closure. Another benefit of linear actuation is thatthe force required for valve actuation may be easily determined from thepressure in the flow channel. If actuation is linear, increased pressurein the flow channel may be countered by adding the same pressure (forceper unit area) to the actuated portion of the valve.

Linearity of a valve depends on the structure, composition, and methodof actuation of the valve structure. Furthermore, whether linearity is adesirable characteristic in a valve depends on the application.Therefore, both linearly and non-linearly actuable valves arecontemplated in the present invention, and the pressure ranges overwhich a valve is linearly actuable will vary with the specificembodiment.

FIG. 9 illustrates time response (i.e.: closure of valve as a functionof time in response to a change in applied pressure) of a 100 μm×100μm×10 μm RTV microvalve with 10-cm-long air tubing connected from thechip to a pneumatic valve as described above.

Two periods of digital control signal, actual air pressure at the end ofthe tubing and valve opening are shown in FIG. 9. The pressure appliedon the control line is 100 kPa, which is substantially higher than the˜40 kPa required to close the valve. Thus, when closing, the valve ispushed closed with a pressure 60 kPa greater than required. Whenopening, however, the valve is driven back to its rest position only byits own spring force (≦40 kPa). Thus, τ_(close) is expected to besmaller than τ_(open). There is also a lag between the control signaland control pressure response, due to the limitations of the miniaturevalve used to control the pressure. Calling such lags t and the 1/e timeconstants τ, the values are: t_(open)=3.63 ms, τ_(open)=1.88 ms,t_(close)=2.15 ms, τ_(close)=0.51 ms. If 3τ each are allowed for openingand closing, the valve runs comfortably at 75 Hz when filled withaqueous solution.

If one used another actuation method which did not suffer from openingand closing lag, this valve would run at ˜375 Hz. Note also that thespring constant can be adjusted by changing the membrane thickness; thisallows optimization for either fast opening or fast closing. The springconstant could also be adjusted by changing the elasticity (Young'smodulus) of the membrane, as is possible by introducing dopant into themembrane or by utilizing a different elastomeric material to serve asthe membrane (described above in conjunction with FIGS. 7C–7H.)

When experimentally measuring the valve properties as illustrated inFIG. 9 the valve opening was measured by fluorescence. In theseexperiments, the flow channel was filled with a solution of fluoresceinisothiocyanate (FITC) in buffer (pH≧8) and the fluorescence of a squarearea occupying the center ˜⅓rd of the channel is monitored on anepi-fluorescence microscope with a photomultiplier tube with a 10 kHzbandwidth. The pressure was monitored with a Wheatstone-bridge pressuresensor (SenSym SCC15GD2) pressurized simultaneously with the controlline through nearly identical pneumatic connections.

6. Flow Channel Cross Sections

The flow channels of the present invention may optionally be designedwith different cross sectional sizes and shapes, offering differentadvantages, depending upon their desired application. For example, thecross sectional shape of the lower flow channel may have a curved uppersurface, either along its entire length or in the region disposed underan upper cross channel). Such a curved upper surface facilitates valvesealing, as follows.

Referring to FIG. 10, a cross sectional view (similar to that of FIG.7B) through flow channels 30 and 32 is shown. As can be seen, flowchannel 30 is rectangular in cross sectional shape. In an alternatepreferred aspect of the invention, as shown in FIG. 10, thecross-section of a flow channel 30 instead has an upper curved surface.

Referring first to FIG. 10, when flow channel 32 is pressurized, themembrane portion 25 of elastomeric block 24 separating flow channels 30and 32 will move downwardly to the successive positions shown by thedotted lines 25A, 25B, 25C, 25D, and 25E. As can be seen, incompletesealing may possibly result at the edges of flow channel 30 adjacentplanar substrate 14.

In the alternate preferred embodiment of FIG. 11, flow channel 30 a hasa curved upper wall 25A. When flow channel 32 is pressurized, membraneportion 25 will move downwardly to the successive positions shown bydotted lines 25A2, 25A3, 25A4 and 25A5, with edge portions of themembrane moving first into the flow channel, followed by top membraneportions. An advantage of having such a curved upper surface at membrane25A is that a more complete seal will be provided when flow channel 32is pressurized. Specifically, the upper wall of the flow channel 30 willprovide a continuous contacting edge against planar substrate 14,thereby avoiding the “island” of contact seen between wall 25 and thebottom of flow channel 30 in FIG. 10.

Another advantage of having a curved upper flow channel surface atmembrane 25A is that the membrane can more readily conform to the shapeand volume of the flow channel in response to actuation. Specifically,where a rectangular flow channel is employed, the entire perimeter (2×flow channel height, plus the flow channel width) must be forced intothe flow channel. However where an arched flow channel is used, asmaller perimeter of material (only the semi-circular arched portion)must be forced into the channel. In this manner, the membrane requiresless change in perimeter for actuation and is therefore more responsiveto an applied actuation force to block the flow channel

In an alternate aspect, (not illustrated), the bottom of flow channel 30is rounded such that its curved surface mates with the curved upper wall25A as seen in FIG. 20 described above.

In summary, the actual conformational change experienced by the membraneupon actuation will depend upon the configuration of the particularelastomeric structure. Specifically, the conformational change willdepend upon the length, width, and thickness profile of the membrane,its attachment to the remainder of the structure, and the height, width,and shape of the flow and control channels and the material propertiesof the elastomer used. The conformational change may also depend uponthe method of actuation, as actuation of the membrane in response to anapplied pressure will vary somewhat from actuation in response to amagnetic or electrostatic force.

Moreover, the desired conformational change in the membrane will alsovary depending upon the particular application for the elastomericstructure. In the simplest embodiments described above, the valve mayeither be open or closed, with metering to control the degree of closureof the valve. In other embodiments however, it may be desirable to alterthe shape of the membrane and/or the flow channel in order to achievemore complex flow regulation. For instance, the flow channel could beprovided with raised protrusions beneath the membrane portion, such thatupon actuation the membrane shuts off only a percentage of the flowthrough the flow channel, with the percentage of flow blockedinsensitive to the applied actuation force.

Many membrane thickness profiles and flow channel cross-sections arecontemplated by the present invention, including rectangular,trapezoidal, circular, ellipsoidal, parabolic, hyperbolic, andpolygonal, as well as sections of the above shapes. More complexcross-sectional shapes, such as the embodiment with protrusionsdiscussed immediately above or an embodiment having concavities in theflow channel, are also contemplated by the present invention.

In addition, while the invention is described primarily above inconjunction with an embodiment wherein the walls and ceiling of the flowchannel are formed from elastomer, and the floor of the channel isformed from an underlying substrate, the present invention is notlimited to this particular orientation. Walls and floors of channelscould also be formed in the underlying substrate, with only the ceilingof the flow channel constructed from elastomer. This elastomer flowchannel ceiling would project downward into the channel in response toan applied actuation force, thereby controlling the flow of materialthrough the flow channel. In general, monolithic elastomer structures asdescribed elsewhere in the instant application are preferred formicrofluidic applications. However, it may be useful to employ channelsformed in the substrate where such an arrangement provides advantages.For instance, a substrate including optical waveguides could beconstructed so that the optical waveguides direct light specifically tothe side of a microfluidic channel.

7. Alternate Valve Actuation Techniques

In addition to pressure based actuation systems described above,optional electrostatic and magnetic actuation systems are alsocontemplated, as follows.

Electrostatic actuation can be accomplished by forming oppositelycharged electrodes (which will tend to attract one another when avoltage differential is applied to them) directly into the monolithicelastomeric structure. For example, referring to FIG. 7B, an optionalfirst electrode 70 (shown in phantom) can be positioned on (or in)membrane 25 and an optional second electrode 72 (also shown in phantom)can be positioned on (or in) planar substrate 14. When electrodes 70 and72 are charged with opposite polarities, an attractive force between thetwo electrodes will cause membrane 25 to deflect downwardly, therebyclosing the “valve” (i.e.: closing flow channel 30).

For the membrane electrode to be sufficiently conductive to supportelectrostatic actuation, but not so mechanically stiff so as to impedethe valve's motion, a sufficiently flexible electrode must be providedin or over membrane 25. Such an electrode may be provided by a thinmetallization layer, doping the polymer with conductive material, ormaking the surface layer out of a conductive material.

In an exemplary aspect, the electrode present at the deflecting membranecan be provided by a thin metallization layer which can be provided, forexample, by sputtering a thin layer of metal such as 20 nm of gold. Inaddition to the formation of a metallized membrane by sputtering, othermetallization approaches such as chemical epitaxy, evaporation,electroplating, and electroless plating are also available. Physicaltransfer of a metal layer to the surface of the elastomer is alsoavailable, for example by evaporating a metal onto a flat substrate towhich it adheres poorly, and then placing the elastomer onto the metaland peeling the metal off of the substrate.

A conductive electrode 70 may also be formed by depositing carbon black(i.e. Cabot Vulcan XC72R) on the elastomer surface, either by wiping onthe dry powder or by exposing the elastomer to a suspension of carbonblack in a solvent which causes swelling of the elastomer, (such as achlorinated solvent in the case of PDMS). Alternatively, the electrode70 may be formed by constructing the entire layer 20 out of elastomerdoped with conductive material (i.e. carbon black or finely dividedmetal particles). Yet further alternatively, the electrode may be formedby electrostatic deposition, or by a chemical reaction that producescarbon. In experiments conducted by the present inventors, conductivitywas shown to increase with carbon black concentration from 5.6×10⁻¹⁶ toabout 5×10⁻³ (Ω-cm)⁻¹. The lower electrode 72, which is not required tomove, may be either a compliant electrode as described above, or aconventional electrode such as evaporated gold, a metal plate, or adoped semiconductor electrode.

Magnetic actuation of the flow channels can be achieved by fabricatingthe membrane separating the flow channels with a magneticallypolarizable material such as iron, or a permanently magnetized materialsuch as polarized NdFeB. In experiments conducted by the presentinventors, magnetic silicone was created by the addition of iron powder(about 1 um particle size), up to 20% iron by weight.

Where the membrane is fabricated with a magnetically polarizablematerial, the membrane can be actuated by attraction in response to anapplied magnetic field Where the membrane is fabricated with a materialcapable of maintaining permanent magnetization, the material can firstbe magnetized by exposure to a sufficiently high magnetic field, andthen actuated either by attraction or repulsion in response to thepolarity of an applied inhomogenous magnetic field.

The magnetic field causing actuation of the membrane can be generated ina variety of ways. In one embodiment, the magnetic field is generated byan extremely small inductive coil formed in or proximate to theelastomer membrane. The actuation effect of such a magnetic coil wouldbe localized, allowing actuation of individual pump and/or valvestructures. Alternatively, the magnetic field could be generated by alarger, more powerful source, in which case actuation would be globaland would actuate multiple pump and/or valve structures at one time.

It is also possible to actuate the device by causing a fluid flow in thecontrol channel based upon the application of thermal energy, either bythermal expansion or by production of gas from liquid. For example, inone alternative embodiment in accordance with the present invention, apocket of fluid (e.g. in a fluid-filled control channel) is positionedover the flow channel. Fluid in the pocket can be in communication witha temperature variation system, for example a heater. Thermal expansionof the fluid, or conversion of material from the liquid to the gasphase, could result in an increase in pressure, closing the adjacentflow channel. Subsequent cooling of the fluid would relieve pressure andpermit the flow channel to open.

8. Networked Systems

FIGS. 12A and 12B show a views of a single on/off valve, identical tothe systems set forth above, (for example in FIG. 7A). FIGS. 13A and 13Bshows a peristaltic pumping system comprised of a plurality of thesingle addressable on/off valves as seen in FIG. 12, but networkedtogether. FIG. 14 is a graph showing experimentally achieved pumpingrates vs. frequency for the peristaltic pumping system of FIG. 13. FIGS.15A and 15B show a schematic view of a plurality of flow channels whichare controllable by a single control line. This system is also comprisedof a plurality of the single addressable on/off valves of FIG. 12,multiplexed together, but in a different arrangement than that of FIG.12. FIG. 16 is a schematic illustration of a multiplexing system adaptedto permit fluid flow through selected channels, comprised of a pluralityof the single on/off valves of FIG. 12, joined or networked together.

Referring first to FIGS. 12A and 12B, a schematic of flow channels 30and 32 is shown. Flow channel 30 preferably has a fluid (or gas) flow Fpassing therethrough. Flow channel 32, (which crosses over flow channel30, as was already explained herein), is pressurized such that membrane25 separating the flow channels may be depressed into the path of flowchannel 30, shutting off the passage of flow F therethrough, as has beenexplained. As such, “flow channel” 32 can also be referred to as a“control line” which actuates a single valve in flow channel 30. InFIGS. 12 to 15, a plurality of such addressable valves are joined ornetworked together in various arrangements to produce pumps, capable ofperistaltic pumping, and other fluidic logic applications.

Referring to FIGS. 13A and 13B, a system for peristaltic pumping isprovided, as follows. A flow channel 30 has a plurality of generallyparallel flow channels (i.e.: control lines) 32A, 32B and 32C passingthereover. By pressurizing control line 32A, flow F through flow channel30 is shut off under membrane 25A at the intersection of control line32A and flow channel 30. Similarly, (but not shown), by pressurizingcontrol line 32B, flow F through flow channel 30 is shut off undermembrane 25B at the intersection of control line 32B and flow channel30, etc.

Each of control lines 32A, 32B, and 32C is separately addressable.Therefore, peristalsis may be actuated by the pattern of actuating 32Aand 32C together, followed by 32A, followed by 32A and 32B together,followed by 32B, followed by 32B and C together, etc. This correspondsto a successive “101, 100, 110, 010, 011, 001” pattern, where “0”indicates “valve open” and “1” indicates “valve closed.” Thisperistaltic pattern is also known as a 120° pattern (referring to thephase angle of actuation between three valves). Other peristalticpatterns are equally possible, including 60° and 90° patterns.

In experiments performed by the inventors, a pumping rate of 2.35 nL/swas measured by measuring the distance traveled by a column of water inthin (0.5 mm i.d.) tubing; with 100×100×10 μm valves under an actuationpressure of 40 kPa. The pumping rate increased with actuation frequencyuntil approximately 75 Hz, and then was nearly constant until above 200Hz. The valves and pumps are also quite durable and the elastomermembrane, control channels, or bond have never been observed to fail. Inexperiments performed by the inventors, none of the valves in theperistaltic pump described herein show any sign of wear or fatigue aftermore than 4 million actuations. In addition to their durability, theyare also gentle. A solution of E. Coli pumped through a channel andtested for viability showed a 94% survival rate.

FIG. 14 is a graph showing experimentally achieved pumping rates vs.frequency for the peristaltic pumping system of FIG. 13.

FIGS. 15A and 15B illustrates another way of assembling a plurality ofthe addressable valves of FIG. 12. Specifically, a plurality of parallelflow channels 30A, 30B, and 30C are provided. Flow channel (i.e.:control line) 32 passes thereover across flow channels 30A, 30B, and30C. Pressurization of control line 32 simultaneously shuts off flowsF1, F2 and F3 by depressing membranes 25A, 25B, and 25C located at theintersections of control line 32 and flow channels 30A, 30B, and 30C.

FIG. 16 is a schematic illustration of a multiplexing system adapted toselectively permit fluid to flow through selected channels, as follows.The downward deflection of membranes separating the respective flowchannels from a control line passing thereabove (for example, membranes25A, 25B, and 25C in FIGS. 15A and 15B) depends strongly upon themembrane dimensions. Accordingly, by varying the widths of flow channelcontrol line 32 in FIGS. 15A and 15B, it is possible to have a controlline pass over multiple flow channels, yet only actuate (i.e.: seal)desired flow channels. FIG. 16 illustrates a schematic of such a system,as follows.

A plurality of parallel flow channels 30A, 30B, 30C, 30D, 30E and 30Fare positioned under a plurality of parallel control lines 32A, 32B,32C, 32D, 32E and 32F. Control channels 32A, 32B, 32C, 32D, 32E and 32Fare adapted to shut off fluid flows F1, F2, F3, F4, F5 and F6 passingthrough parallel flow channels 30A, 30B, 30C, 30D, 30E and 30F using anyof the valving systems described above, with the following modification.

Each of control lines 32A, 32B, 32C, 32D, 32E and 32F have both wide andnarrow portions. For example, control line 32A is wide in locationsdisposed over flow channels 30A, 30C and 30E. Similarly, control line32B is wide in locations disposed over flow channels 30B, 30D and 30F,and control line 32C is wide in locations disposed over flow channels30A, 30B, 30E and 30F.

At the locations where the respective control line is wide, itspressurization will cause the membrane (25) separating the flow channeland the control line to depress significantly into the flow channel,thereby blocking the flow passage therethrough. Conversely, in thelocations where the respective control line is narrow, membrane (25)will also be narrow. Accordingly, the same degree of pressurization willnot result in membrane (25) becoming depressed into the flow channel(30). Therefore, fluid passage thereunder will not be blocked.

For example, when control line 32A is pressurized, it will block flowsF1, F3 and F5 in flow channels 30A, 30C and 30E. Similarly, when controlline 32C is pressurized, it will block flows F1, F2, F5 and F6 in flowchannels 30A, 30B, 30E and 30F. As can be appreciated, more than onecontrol line can be actuated at the same time. For example, controllines 32A and 32C can be pressurized simultaneously to block all fluidflow except F4 (with 32A blocking F1, F3 and F5; and 32C blocking F1,F2, F5 and F6).

By selectively pressurizing different control lines (32) both togetherand in various sequences, a great degree of fluid flow control can beachieved. Moreover, by extending the present system to more than sixparallel flow channels (30) and more than four parallel control lines(32), and by varying the positioning of the wide and narrow regions ofthe control lines, very complex fluid flow control systems may befabricated. A property of such systems is that it is possible to turn onany one flow channel out of n flow channels with only 2(log₂n) controllines.

9. Selectively Addressable Reaction Chambers Along Flow Lines

In a further embodiment of the invention, illustrated in FIGS. 17A, 17B,17C and 17D, a system for selectively directing fluid flow into one moreof a plurality of reaction chambers disposed along a flow line isprovided.

FIG. 17A shows a top view of a flow channel 30 having a plurality ofreaction chambers 80A and 80B disposed therealong. Preferably flowchannel 30 and reaction chambers 80A and 80B are formed together asrecesses into the bottom surface of a first layer 100 of elastomer.

FIG. 17B shows a bottom plan view of another elastomeric layer 110 withtwo control lines 32A and 32B each being generally narrow, but havingwide extending portions 33A and 33B formed as recesses therein.

As seen in the exploded view of FIG. 17C, and assembled view of FIG.17D, elastomeric layer 110 is placed over elastomeric layer 100. Layers100 and 110 are then bonded together, and the integrated system operatesto selectively direct fluid flow F (through flow channel 30) into eitheror both of reaction chambers 80A and 80B, as follows. Pressurization ofcontrol line 32A will cause the membrane 25 (i.e.: the thin portion ofelastomer layer 100 located below extending portion 33A and over regions82A of reaction chamber 80A) to become depressed, thereby shutting offfluid flow passage in regions 82A, effectively sealing reaction chamber80 from flow channel 30. As can also be seen, extending portion 33A iswider than the remainder of control line 32A. As such, pressurization ofcontrol line 32A will not result in control line 32A sealing flowchannel 30.

As can be appreciated, either or both of control lines 32A and 32B canbe actuated at once. When both control lines 32A and 32B are pressurizedtogether, sample flow in flow channel 30 will enter neither of reactionchambers 80A or 80B.

The concept of selectably controlling fluid introduction into variousaddressable reaction chambers disposed along a flow line (FIGS. 17A–D)can be combined with concept of selectably controlling fluid flowthrough one or more of a plurality of parallel flow lines (FIG. 16) toyield a system in which a fluid sample or samples can be can be sent toany particular reaction chamber in an array of reaction chambers. Anexample of such a system is provided in FIG. 18, in which parallelcontrol channels 32A, 32B and 32C with extending portions 34 (all shownin phantom) selectively direct fluid flows F1 and F2 into any of thearray of reaction wells 80A, 80B, 80C or 80D as explained above; whilepressurization of control lines 32C and 32D selectively shuts off flowsF2 and F1, respectively.

In yet another novel embodiment, fluid passage between parallel flowchannels is possible. Referring to FIG. 19, either or both of controllines 32A or 32D can be depressurized such that fluid flow throughlateral passageways 35 (between parallel flow channels 30A and 30B) ispermitted. In this aspect of the invention, pressurization of controllines 32C and 32D would shut flow channel 30A between 35A and 35B, andwould also shut lateral passageways 35B. As such, flow entering as flowF1 would sequentially travel through 30A, 35A and leave 30B as flow F4.

10. Switchable Flow Arrays

In yet another novel embodiment, fluid passage can be selectivelydirected to flow in either of two perpendicular directions. An exampleof such a “switchable flow array” system is provided in FIGS. 20A to20D. FIG. 20A shows a bottom view of a first layer of elastomer 90, (orany other suitable substrate), having a bottom surface with a pattern ofrecesses forming a flow channel grid defined by an array of solid posts92, each having flow channels passing therearound.

In preferred aspects, an additional layer of elastomer is bound to thetop surface of layer 90 such that fluid flow can be selectively directedto move either in direction F1, or perpendicular direction F2. FIG. 20is a bottom view of the bottom surface of the second layer of elastomer95 showing recesses formed in the shape of alternating “vertical”control lines 96 and “horizontal” control lines 94. “Vertical” controllines 96 have the same width therealong, whereas “horizontal” controllines 94 have alternating wide and narrow portions, as shown.

Elastomeric layer 95 is positioned over top of elastomeric layer 90 suchthat “vertical” control lines 96 are positioned over posts 92 as shownin FIG. 20C and “horizontal” control lines 94 are positioned with theirwide portions between posts 92, as shown in FIG. 20D.

As can be seen in FIG. 20C, when “vertical” control lines 96 arepressurized, the membrane of the integrated structure formed by theelastomeric layer initially positioned between layers 90 and 95 inregions 98 will be deflected downwardly over the array of flow channelssuch that flow in only able to pass in flow direction F2 (i.e.:vertically), as shown.

As can be seen in FIG. 20D, when “horizontal” control lines 94 arepressurized, the membrane of the integrated structure formed by theelastomeric layer initially positioned between layers 90 and 95 inregions 99 will be deflected downwardly over the array of flow channels,(but only in the regions where they are widest), such that flow in onlyable to pass in flow direction F1 (i.e.: horizontally), as shown.

The design illustrated in FIG. 20 allows a switchable flow array to beconstructed from only two elastomeric layers, with no vertical viaspassing between control lines in different elastomeric layers required.If all vertical flow control lines 94 are connected, they may bepressurized from one input. The same is true for all horizontal flowcontrol lines 96.

11. Normally-Closed Valve Structure

FIGS. 7B and 7H above depict a valve structure in which the elastomericmembrane is moveable from a first relaxed position to a second actuatedposition in which the flow channel is blocked. However, the presentinvention is not limited to this particular valve configuration.

FIGS. 21A–21J show a variety of views of a normally-closed valvestructure in which the elastomeric membrane is moveable from a firstrelaxed position blocking a flow channel, to a second actuated positionin which the flow channel is open, utilizing a negative controlpressure.

FIG. 21A shows a plan view, and FIG. 21B shows a cross sectional viewalong line 42B–42B′, of normally-closed valve 4200 in an unactuatedstate. Flow channel 4202 and control channel 4204 are formed inelastomeric block 4206 overlying substrate 4205. Flow channel 4202includes a first portion 4202 a and a second portion 4202 b separated byseparating portion 4208. Control channel 4204 overlies separatingportion 4208. As shown in FIG. 42B, in its relaxed, unactuated position,separating portion 4008 remains positioned between flow channel portions4202 a and 4202 b, interrupting flow channel 4202.

FIG. 21C shows a cross-sectional view of valve 4200 wherein separatingportion 4208 is in an actuated position. When the pressure withincontrol channel 4204 is reduced to below the pressure in the flowchannel (for example by vacuum pump), separating portion 4208experiences an actuating force drawing it into control channel 4204. Asa result of this actuation force membrane 4208 projects into controlchannel 4204, thereby removing the obstacle to a flow of materialthrough flow channel 4202 and creating a passageway 4203. Upon elevationof pressure within control channel 4204, separating portion 4208 willassume its natural position, relaxing back into and obstructing flowchannel 4202.

The behavior of the membrane in response to an actuation force may bechanged by varying the width of the overlying control channel.Accordingly, FIGS. 21D–42H show plan and cross-sectional views of analternative embodiment of a normally-closed valve 4201 in which controlchannel 4207 is substantially wider than separating portion 4208. Asshown in cross-sectional views FIGS. 21E–F along line 42E–42E′ of FIG.21D, because a larger area of elastomeric material is required to bemoved during actuation, the actuation force necessary to be applied isreduced.

FIGS. 21G and H show a cross-sectional views along line 40G–40G′ of FIG.21D. In comparison with the unactuated valve configuration shown in FIG.21G, FIG. 21H shows that reduced pressure within wider control channel4207 may under certain circumstances have the unwanted effect of pullingunderlying elastomer 4206 away from substrate 4205, thereby creatingundesirable void 4212.

Accordingly, FIG. 21I shows a plan view, and FIG. 21J shows across-sectional view along line 21J–21J′ of FIG. 21I, of valve structure4220 which avoids this problem by featuring control line 4204 with aminimum width except in segment 4204 a overlapping separating portion4208. As shown in FIG. 21J, even under actuated conditions the narrowercross-section of control channel 4204 reduces the attractive force onthe underlying elastomer material 4206, thereby preventing thiselastomer material from being drawn away from substrate 4205 andcreating an undesirable void.

While a normally-closed valve structure actuated in response to pressureis shown in FIGS. 21A–21J, a normally-closed valve in accordance withthe present invention is not limited to this configuration. For example,the separating portion obstructing the flow channel could alternativelybe manipulated by electric or magnetic fields, as described extensivelyabove.

12. Side-Actuated Valve

While the above description has focused upon microfabricated elastomericvalve structures in which a control channel is positioned above andseparated by an intervening elastomeric membrane from an underlying flowchannel, the present invention is not limited to this configuration.FIGS. 22A and 22B show plan views of one embodiment of a side-actuatedvalve structure in accordance with one embodiment of the presentinvention.

FIG. 22A shows side-actuated valve structure 4800 in an unactuatedposition. Flow channel 4802 is formed in elastomeric layer 4804. Controlchannel 4806 abutting flow channel 4802 is also formed in elastomericlayer 4804. Control channel 4806 is separated from flow channel 4802 byelastomeric membrane portion 4808. A second elastomeric layer (notshown) is bonded over bottom elastomeric layer 4804 to enclose flowchannel 4802 and control channel 4806.

FIG. 22B shows side-actuated valve structure 4800 in an actuatedposition. In response to a build up of pressure within control channel4806, membrane 4808 deforms into flow channel 4802, blocking flowchannel 4802. Upon release of pressure within control channel 4806,membrane 4808 would relax back into control channel 4806 and open flowchannel 4802.

While a side-actuated valve structure actuated in response to pressureis shown in FIGS. 22A and 22B, a side-actuated valve in accordance withthe present invention is not limited to this configuration. For example,the elastomeric membrane portion located between the abutting flow andcontrol channels could alternatively be manipulated by electric ormagnetic fields, as described extensively above.

13. Composite Structures

Microfabricated elastomeric structures of the present invention may becombined with non-elastomeric materials to create composite structures.FIG. 23 shows a cross-sectional view of one embodiment of a compositestructure in accordance with the present invention. FIG. 23 showscomposite valve structure 5700 including first, thin elastomer layer5702 overlying semiconductor-type substrate 5704 having channel 5706formed therein. Second, thicker elastomer layer 5708 overlies firstelastomer layer 5702. Actuation of first elastomer layer 5702 to driveit into channel 5706, will cause composite structure 5700 to operate asa valve.

FIG. 24 shows a cross-sectional view of a variation on this theme,wherein thin elastomer layer 5802 is sandwiched between two hard,semiconductor substrates 5804 and 5806, with lower substrate 5804featuring channel 5808. Again, actuation of thin elastomer layer 5802 todrive it into channel 5808 will cause composite structure 5810 tooperate as a valve.

The structures shown in FIGS. 23 or 24 may be fabricated utilizingeither the multilayer soft lithography or encapsulation techniquesdescribed above. In the multilayer soft lithography method, theelastomer layer(s) would be formed and then placed over thesemiconductor substrate bearing the channel. In the encapsulationmethod, the channel would be first formed in the semiconductorsubstrate, and then the channel would be filled with a sacrificialmaterial such as photoresist. The elastomer would then be formed inplace over the substrate, with removal of the sacrificial materialproducing the channel overlaid by the elastomer membrane. As isdiscussed in detail below in connection with bonding of elastomer toother types of materials, the encapsulation approach may result in astronger seal between the clastomer membrane component and theunderlying nonelastomer substrate component.

As shown in FIGS. 23 and 24, a composite structure in accordance withembodiments of the present invention may include a hard substrate thatbears a passive feature such as a channels. However, the presentinvention is not limited to this approach, and the underlying hardsubstrate may bear active features that interact with an elastomercomponent bearing a recess. This is shown in FIG. 25, wherein compositestructure 5900 includes elastomer component 5902 containing recess 5904having walls 5906 and ceiling 5908. Ceiling 5908 forms flexible membraneportion 5909. Elastomer component 5902 is sealed against substantiallyplanar nonelastomeric component 5910 that includes active device 5912.Active device 5912 may interact with material present in recess 5904and/or flexible membrane portion 5909.

Many Types of active structures may be present in the nonelastomersubstrate. Active structures that could be present in an underlying hardsubstrate include, but are not limited to, resistors, capacitors,photodiodes, transistors, chemical field effect transistors (chemFET's), amperometric/coulometric electrochemical sensors, fiber optics,fiber optic interconnects, light emitting diodes, laser diodes, verticalcavity surface emitting lasers (VCSEL's), micromirrors, accelerometers,pressure sensors, flow sensors, CMOS imaging arrays, CCD cameras,electronic logic, microprocessors, thermistors, Peltier coolers,waveguides, resistive heaters, chemical sensors, strain gauges,inductors, actuators (including electrostatic, magnetic,electromagnetic, bimetallic, piezoelectric, shape-memory-alloy based,and others), coils, magnets, electromagnets, magnetic sensors (such asthose used in hard drives, superconducting quantum interference devices(SQUIDS) and other types), radio frequency sources and receivers,microwave frequency sources and receivers, sources and receivers forother regions of the electromagnetic spectrum, radioactive particlecounters, and electrometers.

As is well known in the art, a vast variety of technologies can beutilized to fabricate active features in semiconductor and other typesof hard substrates, including but not limited printed circuit board(PCB) technology, CMOS, surface micromachining, bulk micromachining,printable polymer electronics, and TFT and otheramorphous/polycrystalline techniques as are employed to fabricate laptopand flat screen displays.

A variety of approaches can be employed to seal the elastomericstructure against the nonelastomeric substrate, ranging from thecreation of a Van der Waals bond between the elastomeric andnonelastomeric components, to creation of covalent or ionic bondsbetween the elastomeric and nonelastomeric components of the compositestructure. Example approaches to sealing the components together arediscussed below, approximately in order of increasing strength.

A first approach is to rely upon the simple hermetic seal resulting fromVan der Waals bonds formed when a substantially planar elastomer layeris placed into contact with a substantially planar layer of a harder,non-elastomer material. In one embodiment, bonding of RTV elastomer to aglass substrate created a composite structure capable of withstanding upto about 3–4 psi of pressure. This may be sufficient for many potentialapplications.

A second approach is to utilize a liquid layer to assist in bonding. Oneexample of this involves bonding elastomer to a hard glass substrate,wherein a weakly acidic solution (5 μl HCl in H₂O, pH 2) was applied toa glass substrate. The elastomer component was then placed into contactwith the glass substrate, and the composite structure baked at 37° C. toremove the water. This resulted in a bond between elastomer andnon-elastomer able to withstand a pressure of about 20 psi. In thiscase, the acid may neutralize silanol groups present on the glasssurface, permitting the elastomer and non-elastomer to enter into goodVan der Waals contact with each other.

Exposure to ethanol can also cause device components to adhere together.In one embodiment, an RTV elastomer material and a glass substrate werewashed with ethanol and then dried under Nitrogen. The RTV elastomer wasthen placed into contact with the glass and the combination baked for 3hours at 80° C. Optionally, the RTV may also be exposed to a vacuum toremove any air bubbles trapped between the slide and the RTV. Thestrength of the adhesion between elastomer and glass using this methodhas withstood pressures in excess of 35 psi. The adhesion created usingthis method is not permanent, and the elastomer may be peeled off of theglass, washed, and resealed against the glass. This ethanol washingapproach can also be employed used to cause successive layers ofelastomer to bond together with sufficient strength to resist a pressureof 30 psi. In alternative embodiments, chemicals such as other alcoholsor diols could be used to promote adhesion between layers.

An embodiment of a method of promoting adhesion between layers of amicrofabricated structure in accordance with the present inventioncomprises exposing a surface of a first component layer to a chemical,exposing a surface of a second component layer to the chemical, andplacing the surface of the first component layer into contact with thesurface of the second elastomer layer.

A third approach is to create a covalent chemical bond between theelastomer component and functional groups introduced onto the surface ofa nonelastomer component. Examples of derivitization of a nonelastomersubstrate surface to produce such functional groups include exposing aglass substrate to agents such as vinyl silane or aminopropyltriethoxysilane (APTES), which may be useful to allow bonding of the glass tosilicone elastomer and polyurethane elastomer materials, respectively.

A fourth approach is to create a covalent chemical bond between theelastomer component and a functional group native to the surface of thenonelastomer component. For example, RTV elastomer can be created withan excess of vinyl groups on its surface. These vinyl groups can becaused to react with corresponding functional groups present on theexterior of a hard substrate material, for example the Si—H bondsprevalent on the surface of a single crystal silicon substrate afterremoval of native oxide by etching. In this example, the strength of thebond created between the elastomer component and the nonelastomercomponent has been observed to exceed the materials strength of theelastomer components.

14. Cell Pen/Cell Cage

In yet a further application of the present invention, an elastomericstructure can be utilized to manipulate organisms or other biologicalmaterial. FIGS. 26A–26D show plan views of one embodiment of a cell penstructure in accordance with the present invention.

Cell pen array 4400 features an array of orthogonally-oriented flowchannels 4402, with an enlarged “pen” structure 4404 at the intersectionof alternating flow channels. Valve 4406 is positioned at the entranceand exit of each pen structure 4404. Peristaltic pump structures 4408are positioned on each horizontal flow channel and on the vertical flowchannels lacking a cell pen structure.

Cell pen array 4400 of FIG. 26A has been loaded with cells A–H that havebeen previously sorted. FIGS. 26B–26C show the accessing and removal ofindividually stored cell C by 1) opening valves 4406 on either side ofadjacent pens 4404 a and 4404 b, 2) pumping horizontal flow channel 4402a to displace cells C and G, and then 3) pumping vertical flow channel4402 b to remove cell C. FIG. 26D shows that second cell G is moved backinto its prior position in cell pen array 4400 by reversing thedirection of liquid flow through horizontal flow channel 4402 a.

The cell pen array 4404 described above is capable of storing materialswithin a selected, addressable position for ready access. However,living organisms such as cells may require a continuous intake of foodsand expulsion of wastes in order to remain viable. Accordingly, FIGS.27A and 27B show plan and cross-sectional views (along line 45B–45B′)respectively, of one embodiment of a cell cage structure in accordancewith the present invention.

Cell cage 4500 is formed as an enlarged portion 4500 a of a flow channel4501 in an elastomeric block 4503 in contact with substrate 4505. Cellcage 4500 is similar to an individual cell pen as described above inFIGS. 26A–26D, except that ends 4500 b and 4500 c of cell cage 4500 donot completely enclose interior region 4500 a. Rather, ends 4500 a and4500 b of cage 4500 are formed by a plurality of retractable pillars4502. Pillars 4502 may be part of a membrane structure of anormally-closed valve structure as described extensively above inconnection with FIGS. 21A–21J.

Specifically, control channel 4504 overlies pillars 4502. When thepressure in control channel 4504 is reduced, elastomeric pillars 4502are drawn upward into control channel 4504, thereby opening end 4500 bof cell cage 4500 and permitting a cell to enter. Upon elevation ofpressure in control channel 4504, pillars 4502 relax downward againstsubstrate 4505 and prevent a cell from exiting cage 4500.

Elastomeric pillars 4502 are of a sufficient size and number to preventmovement of a cell out of cage 4500, but also include gaps 4508 whichallow the flow of nutrients into cage interior 4500 a in order tosustain cell(s) stored therein. Pillars 4502 on opposite end 4500 c aresimilarly configured beneath second control channel 4506 to permitopening of the cage and removal of the cell as desired.

The cross-flow channel architecture illustrated shown in FIGS. 26A–26Dcan be used to perform functions other than the cell pen just described.For example, the cross-flow channel architecture can be utilized inmixing applications.

This is shown in FIGS. 28A–B, which illustrate a plan view of mixingsteps performed by a microfabricated structure in accordance anotherembodiment of the present invention. Specifically, portion 7400 of amicrofabricated mixing structure comprises first flow channel 7402orthogonal to and intersecting with second flow channel 7404. Controlchannels 7406 overlie flow channels 7402 and 7404 and form valve pairs7408 a–b and 7408 c–d that surround each intersection 7412.

As shown in FIG. 28A, valve pair 7408 a–b is initially opened whilevalve pair 7408 c–d is closed, and fluid sample 7410 is flowed tointersection 7412 through flow channel 7402. Valve pair 7408 c–d is thenactuated, trapping fluid sample 7410 at intersection 7412.

Next, as shown in FIG. 28B, valve pairs 7408 a–b and 7408 c–d areopened, such that fluid sample 7410 is injected from intersection 7412into flow channel 7404 bearing a cross-flow of fluid. The process shownin FIGS. 28A–B can be repeated to accurately dispense any number offluid samples down cross-flow channel 7404. FIG. 44 plots Log(R/B) vs.number of slugs injected for one embodiment of a cross-flow injectionsystem in accordance with the present invention. The reproducibility andrelative independence of metering by cross-flow injection from processparameters such as flow resistance is further evidenced by FIG. 82,which plots injected volume versus number of injection cycles forcross-channel flow injection under a variety of flow conditions. FIG. 82shows that volumes metered by cross-flow injection techniques increaseon a linear basis over a succession of injection cycles. This linearrelationship between volume and number of injection cycles is relativelyindependent of flow resistance parameters such as elevated fluidviscosity (imparted by adding 25% glycerol) and the length of the flowchannel (1.0–2.5 cm).

While the embodiment shown and described above in connection with FIGS.28A–28B utilizes linked valve pairs on opposite sides of the flowchannel intersections, this is not required by the present invention.Other configurations, including linking of adjacent valves of anintersection, or independent actuation of each valve surrounding anintersection, are possible to provide the desired flow characteristics.With the independent valve actuation approach however, it should berecognized that separate control structures would be utilized for eachvalve, complicating device layout.

15. Metering By Volume Exclusion

Many high throughput screening and diagnostic applications call foraccurate combination and of different reagents in a reaction chamber.Given that it is frequently necessary to prime the channels of amicrofluidic device in order to ensure fluid flow, it may be difficultto ensure mixed solutions do not become diluted or contaminated by thecontents of the reaction chamber prior to sample introduction.

Volume exclusion is one technique enabling precise metering of theintroduction of fluids into a reaction chamber. In this approach, areaction chamber may be completely or partially emptied prior to sampleinjection. This method reduces contamination from residual contents ofthe chamber contents, and may be used to accurately meter theintroduction of solutions in a reaction chamber.

Specifically, FIGS. 29A–29D show cross-sectional views of a reactionchamber in which volume exclusion is employed to meter reactants. FIG.29A shows a cross-sectional view of portion 6300 of a microfluidicdevice comprising first elastomer layer 6302 overlying second elastomerlayer 6304. First elastomer layer 6302 includes control chamber 6306 influid communication with a control channel (not shown). Control chamber6306 overlies and is separated from dead-end reaction chamber 6308 ofsecond elastomer layer 6304 by membrane 6310. Second elastomer layer6304 further comprises flow channel 6312 leading to dead-end reactionchamber 6308.

FIG. 29B shows the result of a pressure increase within control chamber6306. Specifically, increased control chamber pressure causes membrane6310 to flex downward into reaction chamber 6308, reducing by volume Vthe effective volume of reaction chamber 6308. This in turn excludes anequivalent volume V of reactant from reaction chamber 6308, such thatvolume V of first reactant X is output from flow channel 6312. The exactcorrelation between a pressure increase in control chamber 6306 and thevolume of material output from flow channel 6312 can be preciselycalibrated.

As shown in FIG. 29C, while elevated pressure is maintained withincontrol chamber 6306, volume V′ of second reactant Y is placed intocontact with flow channel 6312 and reaction chamber 6308.

In the next step shown in FIG. 29D, pressure within control chamber 6306is reduced to original levels. As a result, membrane 6310 relaxes andthe effective volume of reaction chamber 6308 increases. Volume V ofsecond reactant Y is sucked into the device. By varying the relativesize of the reaction and control chambers, it is possible to accuratelymix solutions at a specified relative concentration. It is worth notingthat the amount of the second reactant Y that is sucked into the deviceis solely dependent upon the excluded volume V, and is independent ofvolume V′ of Y made available at the opening of the flow channel.

While FIGS. 29A–29D show a simple embodiment of the present inventioninvolving a single reaction chamber, in more complex embodimentsparallel structures of hundreds or thousands of reaction chambers couldbe actuated by a pressure increase in a single control line.

Moreover, while the above description illustrates two reactants beingcombined at a relative concentration that fixed by the size of thecontrol and reaction chambers, a volume exclusion technique could beemployed to combine several reagents at variable concentrations in asingle reaction chamber. One possible approach is to use several,separately addressable control chambers above each reaction chamber. Anexample of this architecture would be to have ten separate control linesinstead of a single control chamber, allowing ten equivalent volumes tobe pushed out or sucked in.

Another possible approach would utilize a single control chamberoverlying the entire reaction chamber, with the effective volume of thereaction chamber modulated by varying the control chamber pressure. Inthis manner, analog control over the effective volume of the reactionchamber is possible. Analog volume control would in turn permit thecombination of many solutions reactants at arbitrary relativeconcentrations.

An embodiment of a method of metering a volume of fluid in accordancewith the present invention comprises providing a chamber having a volumein an elastomeric block separated from a control recess by anelastomeric membrane, and supplying a pressure to the control recesssuch that the membrane is deflected into the chamber and the volume isreduced by a calibrated amount, thereby excluding from the chamber thecalibrated volume of fluid.

II. Crystallization Structures and Methods

High throughput screening of crystallization of a target material, orpurification of small samples of target material by recrystallization,is accomplished by simultaneously introducing a solution of the targetmaterial at known concentrations into a plurality of chambers of amicrofabricated fluidic device. The microfabricated fluidic device isthen manipulated to vary solution conditions in the chambers, therebysimultaneously providing a large number of crystallization environments.Control over changed solvent conditions may result from a variety oftechniques, including but not limited to metering of volumes of acrystallizing agent into the chamber by volume exclusion, by entrapmentof liquid volumes determined by the dimensions of the microfabricatedstructure, or by cross-channel injection into a matrix of junctionsdefined by intersecting orthogonal flow channels.

Crystals resulting from crystallization in accordance with embodimentsof the present invention can be utilized for x-ray crystallography todetermine three-dimensional molecular structure. Alternatively, wherehigh throughput screening in accordance with embodiments of the presentinvention does not produce crystals of sufficient size for direct x-raycrystallography, the crystals can be utilized as seed crystals forfurther crystallization experiments. Promising screening results canalso be utilized as a basis for further screening focusing on a narrowerspectrum of crystallization conditions, in a manner analogous to the useof standardized sparse matrix techniques.

Systems and methods in accordance with embodiments of the presentinvention are particularly suited to crystallizing larger biologicalmacromolecules or aggregates thereof, such as proteins, nucleic acids,viruses, and protein/ligand complexes. However, crystallization inaccordance with the present invention is not limited to any particulartype of target material.

As employed in the following discussion, the term “crystallizing agent”describes a substance that is introduced to a solution of targetmaterial to lessen solubility of the target material and thereby inducecrystal formation. Crystallizing agents typically includecountersolvents in which the target exhibits reduced solubility, but mayalso describe materials affecting solution pH or materials such aspolyethylene glycol that effectively reduce the volume of solventavailable to the target material. The term “countersolvent” is usedinterchangeably with “crystallizing agent”.

1. Crystallization by Volume Exclusion

FIG. 30 shows a plan view of one embodiment of a crystallization systemthat allows mass crystallization attempts employing the volume exclusiontechnique described in conjunction with prior FIGS. 29A–D.

Crystallization system 7200 comprises control channel 7202 and flowchannels 7204 a, 7204 b, 7204 c, and 7204 d. Each of flow channels 7204a, 7204 b, 7204 c, and 7204 d feature dead-end chambers 7206 that serveas the site for crystallization. Control channel 7202 features a networkof control chambers 7205 of varying widths that overlie and areseparated from chambers 7206 by membranes 7208 having the same widths ascontrol chambers 7205. Although not shown to clarify the drawing, asecond control featuring a second network of membranes may be utilizedto create stop valves for selectively opening and closing the openingsto dead-end chambers 7206. A full discussion of the function and role ofsuch stop valves is provided below in conjunction with FIG. 31.

Operation of crystallization system 7200 is as follows. Initially, anaqueous solution containing the target protein is flushed through eachof flow channels 7204 a, 7204 b, 7204 c, and 7204 d, filling eachdead-end chamber 7206. Next, a high pressure is applied to controlchannel 7202 to deflect membranes 7208 into the underlying chambers7206, excluding a given volume from chamber 7206 and flushing thisexcluded volume of the original protein solution out of chamber 7206.

Next, while pressure is maintained in control channel 7202, a differentcountersolvent is flowed into each flow channel 7204 a, 7204 b, 7204 c,and 7204 d. Pressure is then released in control line 7202, andmembranes 7208 relax back into their original position, permitting theformerly excluded volume of countersolvent to enter chambers 7206 andmix with the original protein solution. Because of the differing widthsof control chambers 7205 and underlying membranes 7208, a variety ofvolumes of the countersolvent enters into chambers 7206 during thisprocess.

For example, chambers 7206 a in the first two rows of system 7200 do notreceive any countersolvent because no volume is excluded by an overlyingmembrane. Chambers 7106 b in the second two rows of system 7200 receivea volume of countersolvent that is 1:5 with the original proteinsolution. Chambers 7206 c in the third two rows of system 7200 receive avolume of countersolvent that is 1:3 with the original protein solution.Chambers 7206 d in the fourth two rows of system 7200 receive a volumeof countersolvent that is 1:2 with the original protein solution, andchambers 7206 e in the fifth two rows of system 7200 receive a volume ofcountersolvent that is 4:5 with the original protein solution.

Once the countersolvent has been introduced into the chambers 7206, theymay be resealed against the environment by again applying a highpressure to control line 7202 to deflect the membranes into thechambers. Resealing may be necessary given that crystallization canrequire on the order of days or weeks to occur. Where visual inspectionof a chamber reveals the presence of a high quality crystal, the crystalmay be physically removed from the chamber of the disposable elastomersystem.

2. Crystallization by Volume Entrapment

While the above description has described a crystallization system thatrelies upon volume exclusion to meter varying amounts of countersolvent,the invention is not limited to this particular embodiment. Accordingly,FIG. 31 shows a plan view of an alternative crystallization systemwherein metering of different volumes of countersolvent is determined byphotolithography during formation of the flow channels.

Crystallization system 7500 comprises flow channels 7504 a, 7504 b, 7504c, and 7504 d. Each of flow channels 7504 a, 7504 b, 7504 c, and 7504 dfeature dead-end chambers 7506 that serve as the site forrecrystallization.

System 7500 further comprises two sets of control channels. First set7502 of control channels overlie the opening of chambers 7506 and definestop valves 7503 that, when actuated, block access to chambers 7506.Second control channels 7505 overlie flow channels 7504 a–d and definesegment valves 7507 that, when actuated, block flow between differentsegments 7514 of a flow channel 7404.

Operation of crystallization system 7500 is as follows. Initially, anaqueous solution containing the target protein is flushed through eachof flow channels 7504 a, 7504 b, 7504 c, and 7504 d, filling dead-endchambers 7506. Next, a high pressure is applied to control channel 7502to actuate stop valves 7503, thereby preventing fluid from entering orexiting chambers 7506.

While maintaining stop valves 7503 closed, each flow channel 7504 a–d isthen filled with a different countersolvent. Next, second control line7505 is pressurized, isolating flow channels 7504 a–d into segments 7514and trapping differing volumes of countersolvent. Specifically, as shownin FIG. 31 segments 7514 are of unequal volumes. During formation ofprotein crystallization structure 7500 by soft lithography,photolithographic techniques are employed to define flow channels 7504a–d having segments 7514 of different widths 7514 a and lengths 7514 b.

Thus, when pressure is released from first control line 7502 and stopvalves 7503 open, a different volume of countersolvent from the varioussegments 7514 may diffuse into chambers 7506. In this manner, precisedimensions defined by photolithography can be employed to determine thevolume of countersolvent trapped in the flow channel segments and thenintroduced to the protein solution. This volume of countersolvent inturn establishes the environment for crystallization of the protein.

While the crystallization system described in connection with FIG. 31utilizes the dimensions of the flow channels to dictate the volumes ofcountersolvents introduced into the crystallization chamber, the presentinvention is not limited to this approach.

FIG. 32 shows a microfabricated crystallization system wherein thevolumes of countersolvent metered to the recrystallization chambers isdictated by the angle of orientation of a control channel relative tounderlying flow channels. Specifically, microfabricated crystallizationsystem 8000 includes adjacent serpentine flow channels 8002 a and 8002 bconnected through a series of bridging channels 8004. First control line8006 overlies bridging channels 8004 and thereby forms valves 8008isolating serpentine channels 8002 a and 8002 b from each other. Secondcontrol line 8010 includes projections over portions of first serpentinechannel 8002 a defining valves 8020.

Initially, first control line 8006 is closed while second and thirdcontrol lines 8010 and 8012 remain open. First serpentine channel 8002 ais filled with target material solution through inlet 8014. While firstserpentine channel 8002 a of FIG. 31 is depicted as having an outlet8015, channel 8002 a may also be dead-ended. Second serpentine channel8002 b is filled with a countersolvent to be mixed with the targetmaterial solution. As with first serpentine channel 8002 a, secondserpentine channel 8002 b may also terminate at an outlet or a dead end.

Next, second control channel 8010 is activated to close valves 8020,thereby isolating equal volumes of target solution trapped in region8022. Third control channel 8012 is also activated to close valves 8024,thereby isolating countersolvent trapped in region 8026 b. However,because third control channel 8012 runs obliquely across secondserpentine channel 8002 b, the volumes of countersolvent entrappedbetween valves 8008 and 8024 is unequal and becomes progressivelysmaller.

Next, first control channel 8006 is activated and valves 8008 opened.The volumes of countersolvent entrapped in region 8026 are now free todiffuse into the volume of sample entrapped in region 8022, with therespective ratios of mixing determined by the relative angularorientation of third control channel 8012.

The crystallization system of FIG. 32 permits one type of countersolventto be introduced to the sample through a single serpentine channel.However, in order to facilitate high throughput crystallizationconditions, a series of crystallization systems as shown in FIG. 32sharing a common sample source could be fabricated on a substrate, withdifferent countersolvent provided to each system.

Moreover, other variations of crystallization system embodimentutilizing metering of countersolvent volume by entrapment are alsopossible. For example, in one alternative embodiment the relativevolumes of a sample could be determined by the angle of orientation ofthe second control channel overlying the samples. Moreover, the shape ofthe flow channels on either side of the bridging channels could bemodified to provide additional volume between successive valves. Otherlithographically determined dimensions such as flow channel depth andwidth could also be controlled to affect the relative volumes ofcountersolvent and sample.

FIG. 43 is a plan view of an alternative embodiment of arecrystallization system in accordance with the present inventionutilizing volume entrapment. The system of FIG. 43 is similar to thatshown in FIG. 31, except that it includes a larger number of flowchannels, along with several features that enhance control over the flowof materials down those channels.

Crystallization system 9000 comprises flow channels 9004 a, 9004 b, 9004c, 9004 d, 9004 e, 9004 f, 9004 g, and 9004 h. Each of flow channels9004 a–h feature dead-end chambers 9006 that serve as the site forrecrystallization. The protein crystallization structure illustrated inthis embodiment of the present invention may utilize less than 1 μL ofsample while creating 64 1 nL recrystallization environments.

The flow of materials down each flow channel 9004 a–h is controlled byseveral valve and pump structures. An initial set of gate valves 9024 isformed by the overlap of gate control channel 9026 over the upstreamportions of respective flow channels 9004 a–h.

A first set 9002 of control channels overlie the opening of chambers9006 and define stop valves 9003 that, when actuated, block access tochambers 9006. Second control channels 9005 overlie flow channels 9004a–h and define segment valves 9007 that, when actuated, block flowbetween different segments 9014 of a flow channel 9004.

The operation of system 9000 is similar to that described above for thesystem of FIG. 31, with charging of chambers and segments with a samplevolume, followed by introduction of volumes of countersolvent. However,Rather than utilizing a simultaneous flow of samples or countersolventsthrough all of the flow channels 8604, system 9000 includes multiplexerstructure 9020 on the output side of the flow channel. Specifically,peristaltic pumping control channels 9020 a–f of varying widths overliethe downstream ends of each of flow channels 9004 a–h. By selectmanipulation of the pressure within pumping control channels 9020 a–f,sample or countersolvents may be independently flowed down each of flowchannels 9004 a–h.

The enhanced precision in control over the flow of materials down theflow channels of the system offers a number of benefits. One benefit isreduced risk of cross-contamination. Because the flow channels areindependently controlled and are in contact with one another onlydownstream of multiplexer structure 9020, incidental pressuredifferences arising between flow channels will not result in unwantedbackflow of material between flow channels.

3. Crystallization by Cross-Channel Injection

The cross-flow channel architecture illustrated in prior FIGS. 26A–26Dcan be used to perform high throughput crystallization of a targetmaterial. This approach is shown in FIG. 33, which illustrates analternative embodiment of a crystallization structure in accordance withthe present invention.

The microfabricated cross-channel high throughput crystallizationstructure of FIG. 33 comprises a five-by-five array 8100 ofcross-injection junctions 8102 formed by the intersection of parallelhorizontal flow channels 8104 and parallel vertical flow channels 8106.Array 8100 enables the mixing and storage of each sample S1–S5 with eachcountersolvent C1–C5, for a total of 5×5=25 simultaneous crystallizationconditions. Movement of the fluid along horizontal flow channels 8104 iscontrolled in parallel by peristaltic pumps 8108 formed by overlyingcontrol channels 8110. Movement of fluid along vertical flow channels8106 is controlled in parallel by peristatic pump 8112 formed byoverlying control channels 8114. As shown in prior FIGS. 28A–B, columnvalves 8116 and row valves 8118 surround each junction 8102 formed bythe intersection of horizontal and vertical flow lines 8104 and 8106.

Column valves 8116 blocking flow in the vertical direction arecontrolled by a single control line 8120. Row valves 8110 blocking flowin the horizontal direction are controlled a single control line 8122.For purposes of illustration, only the first portion of control lines8120 and 8122 are shown in FIG. 33, it is to be understood that everyrow and column valve is controlled by these control lines.

During crystallization, horizontal flow channels 8104 introduce samplesof five different concentrations of target material to junctions 8102,while vertical flow channels 8106 introduce to junctions 8102 fivedifferent concentrations and/or compositions of countersolvent. Throughthe metering technique described below in connection with FIGS. 34A–34C,all 5×5=25 possible combinations of sample and countersolvent are storedat the 5×5=25 junctions 8102 of array 8100.

FIGS. 34A–34C show enlarged plan views of adjacent junctions of array8100 of FIG. 32. For purposes of illustration, the control lines areomitted in FIGS. 34A–34C. Also, the lateral distance between junctionsis considerably shortened, and in actuality the junctions would beseparated by a considerable distance to prevent cross-contamination.

In a first step shown in FIG. 34A, column valves 8116 are closed and asample of target material at a given concentration is flowed down firsteach of horizontal flow channels 8104. In the array portion shownenlarged in FIG. 34A, inter-row valve regions 8126 are thereby chargedwith sample material S1.

Next, as shown in FIG. 34B, row valves 8118 are closed, and columnvalves 8116 are opened. Countersolvents of different concentrationsand/or compositions are flowed down each of vertical flow channels 8106.In the array portion enlarged in FIG. 34B, junctions 8102 are therebycharged with countersolvents C1 and C2.

As shown in FIG. 34C, column valves 8116 are closed and row valves 8118are opened. Pumping of the peripheral peristaltic pumps of the arraycauses the sample in inter-valve regions 8126 to mingle withcountersolvent of junctions 8102 as both are flowed into junctions 8102and inter-valve regions 8126. Row valves 8118 are then closed as columnvalves 8116 are maintained closed to prevent cross-contamination betweencrystallization sites. In the array portion enlarged in FIG. 34C,crystallization may then take place in solvent environments S1C1 andS1C2.

In an alternative embodiment of the present invention, separate controllines could be used to control actuation of alternate row valves. Insuch an embodiment, once the inter-row valve regions and the junctionshave been charged with sample and countersolvent as described above inFIGS. 34A and 34B, in the third step the alternate row valves are openedsuch that sample in inter-row valve regions mixes by diffusion withcountersolvent in junctions. This alternative embodiment does notrequire pumping, and the closed state of the other set of alternate rowvalves prevents cross-contamination.

In yet another alternative embodiment of a structure for performinghigh-throughput crystallization screening in accordance with the presentinvention, a single control line may be utilized to control alternaterow valves to define a plurality of crystallization screening chambers.This embodiment is shown in FIGS. 81A–81B.

FIG. 81A shows an enlarged view of a portion of one flow channel inaccordance with an alternative embodiment of the present invention. FIG.81B shows cross-sectional view along line B–B′ of the enlarged flowchannel portion of FIG. 81A prior to deactuation of alternative rowvalves to allow diffusion between crystallizing agent and targetmaterial in adjacent chambers. FIG. 81C shows cross-sectional view alongline B–B′ of the enlarged flow channel portion of FIG. 81A afterdeactuation of alternative row valves to allow diffusion betweencrystallizing agent and target material in adjacent chambers.

Control line 8150 comprises parallel branches 8150 a and 8150 bpositioned on either side of flow channel 8104. Branches 8150 a–b areconnected by alternating wide cross-over portions 8152 and narrowcross-over portions 8154, thereby defining large valves 8156 and smallvalves 8158, respectively. Because of the differing width of cross-overportions 8152 and 8154, elastomer membranes 8160 a and 8160 b of valves8156 and 8158 are deactuated and actuated, respectively, when differentpressures are applied to control line 8150.

Specifically, application of a highest pressure to control line 8150will cause the deflection of both elastomer membranes 8160 a and 8160 binto the underlying flow channel 8104, closing both large valves 8156and small valves 8158. Application of a lowest pressure to control line8150 will cause both elastomer membranes 8160 a and 8160 b to relax outof the underlying flow channel 8104, opening both large valves 8156 andsmall valves 8158.

Due to their increased area, wide membranes 8160 a are easier to actuatethan narrow membranes 8160 b. Accordingly, application of anintermediate pressure to control line 8150 will result in only widemembranes 8160 a of large valve structures 8156 remaining actuated, withnarrow membranes 8160 b of small valve structures 8158 being deactuatedto open the valve. This differential response to an applied pressure canallow the use of only one control line to define a plurality ofcrystallization screening chambers, and then to allow alternative valvesalong a horizontal flow channel to relax, permitting diffusion of targetmaterial and crystallizing agent.

During operation of the microfluidic structure shown in FIGS. 81A–C, asdescribed above in connection with FIGS. 34A–B, in FIGS. 81A–B, flowchannel junctions 8102 are filled with crystallizing agent andinter-valve regions 8126 are filled with target sample by applying highand low pressures to the control line 8150.

As shown in FIG. 81C, when an intermediate pressure is applied tocontrol line 8150, narrow membranes 8160 a of small valve structures8158 to relax out of flow channel 8104, while wide membranes 8160 a oflarge valve structures 8156 remain within flow channel 8104 to preventcross-contamination between adjacent crystallization sites.

4. Crystallization Utilizing Diffusion/Dialysis

One conventional approach to crystallization has been to effect agradual change in target solution conditions by introducing acrystallizing agent through slow diffusion, or slow diffusion inconjunction with dialysis. For example, in the crystallization ofproteins, imposing a dialysis membrane between sample and crystallizingagent results in diffusion of crystallizing agent into the proteinsolution without reduction in concentration of the protein sample.

Crystallization methods and structures in accordance with embodiments ofthe present invention utilizing slow diffusion and/or dialysis mayemploy a variety of techniques. Several possible approaches aredescribed below.

In a first embodiment shown in FIG. 35, microfabricated elastomericstructure 8200 features chambers 8202 of varying volumes that may beinitially charged with samples through pump/valve network. Chambers 8202are also in fluid communication with face 8200 a of structure 8200.Dialysis membrane 8204 is fixed to face 8200 a, and then the entiremicrofabricated structure 8200 is immersed in bulk countersolventreservoir 8200 as shown. Over time, countersolvent from reservoir 8206diffuses across membrane 8204 and into chambers 8202 and solvent fromthe sample diffuses across membrane 8204 into reservoir 8206. Protein ofthe sample is prevented from diffusing by membrane 8204. When thedesired solution conditions are achieved, a crystal may form in chamber8202.

The advantage of this approach to crystallization is simplicity, in thatonce charged with sample, the microfabricated elastomeric structure issimply dunked in the countersolvent. This approach also enables directmonitoring of solution conditions, as the pH, temperature, and otheraspects of the bulk countersolvent reservoir can be monitored forchanges using conventional detection methods. Moreover, in alternativeembodiments of the present invention, a continuous supply of dissolvedtarget material may be flowed past the dialysis membrane to ensure anadequate supply for growth of large crystals.

Embodiments in accordance with the present invention may also beimplemented in conjunction with double dialysis, wherein rate of changein condition of the target solution is slowed by imposing a seconddialysis membrane and an intermediate solution between the crystallizingagent and the first dialysis membrane. In such an approach, theintermediate solution serves to buffer changes in the target solutionarising from diffusion of crystallizing agent. In the technique justdescribed, double dialysis could be accomplished by immersing themicrofluidic structure and the associated dialysis membrane in anintermediate solution in fluid communication with a crystallizing agentreservoir through a second dialysis membrane.

A second embodiment of the present invention employing dialysistechniques is illustrated in FIG. 36. This approach utilizes dialysismembrane 8300 sandwiched between opposing microfabricated elastomericstructures 8302 and 8304. Upon assembly of this structure and properalignment of respective chambers/channels 8306 of opposing structures8302 and 8304, countersolvent from reservoirs 8308 of structure 8302will diffuse across membrane 8300 into the correspondingrecrystallization chamber 8310 of structure 8304. Solvent fromcrystallization chamber 8310 will correspondingly diffuse acrossmembrane 8300 into reservoir 8308 of first structure 8302. However,protein in crystallization chamber 8310 will be prevented by membrane8300 from similarly diffusing, and will thus be retained in chamber 8310as the solution environment is changed.

Double dialysis employing a structure similar to that of FIG. 36 couldbe accomplished by fabricating an intermediate chamber between thecrystallization chamber and the first dialysis membrane, and thenfilling this intermediate chamber with a buffer solution. A seconddialysis membrane could be introduced into the microfabricated structurebetween the intermediate and crystallization chambers in the form of aplug of a cross-linked polymer, as described below in FIG. 37.

The embodiments just described in FIGS. 35 and 36 utilize large scalebonding of a dialysis membrane to an entire face of a microfabricatedstructure. However, other embodiments may utilize the insertion orplacement of a dialysis membrane within local regions of amicrofabricated structure. This is shown in FIG. 36, wherein a dialysismembrane is created within the microfabricated structure in the form ofa polyacrylamide gel.

Specifically, recrystallization structure 8400 of FIG. 37 includes firstchamber 8402 in fluid communication with dead-ended chamber 8404 throughhorizontal flow channel 8406. The intersection of horizontal flowchannel 8406 and vertical flow channel 8408 creates junction 8410. Firstvalve set 8412 is defined by the overlap of first control channel 8414and portions 8416 a of horizontal flow channel 8406 on opposite sides ofjunction 8410. Second valve set 8416 is defined by the overlap of secondcontrol channel 8418 and portions 8408 a of vertical flow channel 8408on opposite sides of junction 8410.

Operation of this embodiment is as follows. Second valve set 8416 isclosed while first valve set 8412 is opened. Dead-ended chamber 8404 ischarged with a sample through horizontal flow channel 8406.

Next, second valve set 8416 is opened and first valve set 8412 isclosed. Vertical flow channel 8408 is charged with a cross-linkablepolymer 8420 such as a polyacrylamide gel. Cross-linking of the polymerwithin vertical flow channel is then induced, for example by irradiationof the flow channel or by mixing slow acting cross-linking chemicalswith the polymer prior or during charging of the vertical flow channelwith gel. Once the desired amount of cross-linking of the polymer hasoccurred, it will serve as a selective barrier to diffusion (i.e. as adialysis membrane).

Finally, second valve set 8416 is closed and first valve set 8412 isagain opened, and first chamber 8402 is charged with countersolvent.This countersolvent diffuses across cross-linked polymer membrane 8420to alter the solution conditions in dead-ended chamber 8404.

Double dialysis to further mediate change in target material solutionconditions over time, could be effected by introducing a microfabricatedchamber and second polyacrylamide plug intermediate to thecrystallization chamber and the chamber containing the crystallizingagent.

In any of the embodiments of double dialysis described above, the seconddialysis membrane could be eliminated, and diffusion of crystallizingagent across the intermediate solution relied upon to slow changes incondition of the target material solution. Diffusion rates of thecrystallizing agent across the intermediate solution could be controlledby the physical dimensions (i.e. length, cross-section) of theintervening structure, such as a microfabricated chamber/channel or acapillary or larger diameter tube connecting reservoirs in whichmicrofabricated structure has been immersed.

In other embodiments, a microfabricated elastomer structure may besliced vertically, often preferably along a channel cross section. Inaccordance with embodiments of the present invention, a non-elastomercomponent may be inserted into the elastomer structure that has beenopened by such a cut, with the elastomer structure then resealed. Oneexample of such an approach is shown in FIGS. 38A–38C, which illustratescross-sectional views of a process for forming a flow channel having amembrane positioned therein. Specifically, FIG. 38A shows across-section of a portion of device 6200 including elastomer membrane6202 overlying flow channel 6204, and elastomer substrate 6206.

FIG. 38B shows the results of cutting device 6200 along vertical line6208 extending along the length of flow channel 6204, such that halves6200 a and 6200 b are formed. FIG. 38C shows insertion of permeablemembrane element 6210 between halves 6200 a and 6200 b, followed byattachment of halves 6200 a and 6200 b to permeable membrane 6210. As aresult of this configuration, the flow channel of the device actuallycomprises channel portions 6204 a and 6204 b separated by permeablemembrane 6210.

The structure of FIG. 38C could be utilized in a variety ofapplications. For example, the membrane could be used to performdialysis, altering the salt concentration of samples in the flowchannel. This would result in a change of the solution environment of acrystallized target material.

While embodiments of the present invention discussed so far utilizediffusion of crystallizing agent in the liquid phase, vapor diffusion isanother technique that has been employed to induce crystal formation.Accordingly, FIGS. 39–41 show a plan view of several embodiments ofvapor diffusion structures in accordance with embodiments of the presentinvention.

FIG. 39 shows a simple embodiment of a vapor diffusion structure 8600,wherein first microfabricated chamber 8602 having inlet 8602 a andoutlet 8602 b and second microfabricated chamber 8604 having inlet 8604a and outlet 8604 b are connected by cross flow channel 8606. Initially,the entire structure 8600 is filled with air. Cross-valves 8608 are thenactuated to trap air within cross-flow channel 8606. Target solution isthen introduced to first chamber 8602 through inlet 8602 a, withdisplaced air escaping through outlet 8602 b. Crystallizing agent isintroduced to second chamber 8604 through inlet 8604 a, with displacedair escaping through outlet 8604 b.

Cross-valves 8608 are then opened, such that air remains trapped withincross-flow channel 8606 between sample and crystallizing agent. Vapordiffusion of solvent and crystallizing agent may then slowly take placeacross the air pocket of cross-flow channel 8606 to change the solutionconditions and thereby induce crystal formation in first chamber 8602.Structure 8600 may be sealed against the outside environment by valves8610 during this process.

While the above embodiment is functional, the air pocket trapped betweenthe liquid-filled chambers may move or deform in response toenvironmental conditions, permitting unwanted direct fluid contactbetween target material solution and crystallizing agent. It istherefore desirable to anchor the air pocket at specific locationswithin the microfabricated structure.

Accordingly, FIG. 40 shows an alternative embodiment of a structure forperforming crystallization by vapor diffusion. Specifically, structure8700 comprises chamber 8702 having first inlet 8704 at a first end 8702a, second inlet 8706 at a second end 8702 b, and vent 8707 at middleportion 8702 c. Middle portion 8702 c of chamber 8702 includeshydrophobic region 8708 which may be formed by microcontact printing.Microcontact printing techniques are described in detail by Andersson etal., “Consecutive Microcontact Printing—Ligands for Asymmetric Catalysisin Silicon Channels”, Sensors and Actuators B, 3997 pp. 1–7 (2001),hereby incorporated by reference for all purposes.

Specifically, during fabrication of structure 8700, the underlyingsubstrate may be stamped with pattern 8710 of octadecyltrichlorosilane(OTS). Subsequent alignment of microfabricated elastomeric chamber 8702over pattern 8710 would form central hydrophobic region 8712.

Initially, structure 8700 would be filled with air. Aqueous targetsolution would then carefully be introduced through first inlet 8706,with air displaced from chamber 8702 through vent 8707. Because of thepresence of hydrophobic chamber region 8712, filling of chamber 8702with target solution would halt as the solution encountered region 8712.Similarly, hydrophilic crystallizing agent would carefully be introducedthrough second inlet 8708 to chamber 8702, stopping at hydrophobicregion 8712. Air displaced by filling of chamber 8702 with crystallizingagent would exit chamber 8702 through vent 8707. Thus secured in placeby the underlying patterned hydrophobic region 8712, the air pocket incentral region 8712 would permit slow vapor diffusion of crystallizingagent into target sample to induce crystal formation on the right sideof chamber 8702. Surrounding valves 8714 could be actuated to isolatethe structure during this process.

While useful, the embodiment of a vapor diffusion structure justdescribed in conjunction with FIG. 40 requires alignment of themicrofabricated elastomeric channel to a patterned hydrophobic region onan underlying substrate. This alignment process may be difficult giventhe small feature sizes of structures in accordance with embodiments ofthe present invention. Moreover, during the fabrication process thehydrophobic material would likely be formed only on underlyingsubstrate, and not on the channel walls.

Accordingly, FIG. 41 shows still another embodiment of a structure forperforming crystallization of target materials by vapor diffusion, whichdoes not require an alignment step. Specifically, recrystallizationstructure 8800 includes first chamber 8802 connected to second chamber8804 by cross-flow channel 8806. Second flow channel 8808 intersectswith cross-flow channel 8806, forming junction 8810. Flow acrossjunction 8810 along cross-flow channel 8806 is controlled by first valvepair 8812. Flow across junction 8810 along second flow channel 8808 iscontrolled by second valve pair 8814.

Initially, first chamber 8802 is charged with target material solutionand second chamber 8804 is charged with crystallizing agent. Next, firstvalve pair 8812 is closed and second valve pair 8814 is opened, andhydrophobic material such as OTS is flowed down second flow channel 8808through junction 8810. As a result of this flow of material, hydrophobicresidue 8816 remains on the substrate and possibly on the flow channelwalls injunction 8810.

Next, air is introduced into second flow channel 8808, and second valvepair 8814 is closed. First valve pair 8812 is then opened to permitvapor diffusion of crystallizing agent in chamber 8804 across air-filledjunction 8810 into target material solution in chamber 8802. During thisvapor diffusion process, the air pocket is fixed in junction 8810 byclosed valve pair 8814 and the presence of hydrophobic residue 8816.Valves 8818 could be closed to completely seal structure 8800 againstthe outside environment.

While the above embodiment has focused upon microcontact printing ofhydrophobic moieties to fix in place air pockets during vapor diffusion,the present invention is not limited to this approach. Hydrophobicregions selectively introduced into portions of a microfabricatedcrystallization structure in accordance with the present invention couldalternatively be utilized to fix in place barriers or impediments todiffusion in the form of hydrophobic oils.

Hydrophobic oil materials may also be utilized to coat the exteriorsurface of microfabricated elastomer structures in accordance withembodiments of the present invention. Such a coating may be impermeableto outdiffusion of vapor from the elastomer, thereby preventingdehydration of the structure during the potentially long crystallizationdurations. Alternatively, the coating oil may be somewhat permeable towater or other gases, thereby allowing for slow, controlled outdiffusionof water or gases to create within the structure conditions favorable tocrystallization.

5. Control Over Other Factors Influencing Crystallization

While the above crystallization structures describe altering theenvironment of the target material through introduction of volumes of anappropriate crystallization agent, many other factors are relevant tocrystallization. Such additional factors include, but are not limitedto, temperature, pressure, concentration of target material in solution,equilibration dynamics, and the presence of seed materials.

In specific embodiments of the present invention, control overtemperature during crystallization may be accomplished utilizing acomposite elastomer/silicon structure previously described.Specifically, a Peltier temperature control structure may be fabricatedin an underlying silicon substrate, with the elastomer aligned to thesilicon such that a crystallization chamber is proximate to the Peltierdevice. Application of voltage of an appropriate polarity and magnitudeto the Peltier device may control the temperature of solvent andcountersolvent within the chamber.

Alternatively, as described by Wu et al. in “MEMS Flow Sensors forNano-fluidic Applications”, Sensors and Actuators A 89 152–158 (2001),crystallization chambers could be heated and cooled through theselective application of current to a micromachined resistor structureresulting in ohmic heating. Moreover, the temperature of crystallizationcould be detected by monitoring the resistance of the heater over time.The Wu et al. paper is hereby incorporated by reference for allpurposes.

It may also be useful to establish a temperature gradient across amicrofabricated elastomeric crystallization structure in accordance withthe present invention. Such a temperature gradient would subject targetmaterials to a broad spectrum of temperatures during crystallization,allowing for extremely precise determination of optimum temperatures forcrystallization.

With regard to controlling pressure during crystallization, embodimentsof the present invention employing metering of countersolvent by volumeexclusion are particularly advantageous. Specifically, once the chamberhas been charged with appropriate volumes of solvent and countersolvent,a chamber inlet valve may be maintained shut while the membraneoverlying the chamber is actuated, thereby causing pressure to increasein the chamber. Structures in accordance with the present inventionemploying techniques other than volume exclusion could exert pressurecontrol by including flow channels and associated membranes adjacent tothe crystallization chamber and specifically relegated to controllingpressure within the channel.

Another factor influencing crystallization is the amount of targetmaterial available in the solution. As a crystal forms, it acts as asink to target material available in solution, to the point where theamount of target material remaining in solution may be inadequate tosustain continued crystal growth. Therefore, in order to growsufficiently large crystals it may be necessary to provide additionaltarget material during the crystallization process.

Accordingly, the cell pen structure previously described in connectionwith FIGS. 27A–27B may be advantageously employed in crystallizationstructures in accordance with embodiments of the present invention toconfine growing crystals within a chamber. This obviates the danger ofwashing growing crystals down a flow channel that is providingadditional target material, causing the growing crystals to be lost inthe waste.

Moreover, the cell cage structure of FIGS. 27A–27B may also be usefulduring the process of crystal identification. Specifically, salts areoften present in the sample or countersolvent, and these salts may formcrystals during crystallization attempts. One popular method ofdistinguishing the growth of salt crystals from the target crystals ofinterest is through exposure to a staining dye such as IZIT™,manufactured by Hampton Research of Laguna Niguel, Calif. This IZIT™ dyestains protein crystals blue, but does not stain salt crystals.

However, in the process of flowing the IZIT™ dye to the crystallizationchamber holding the crystals, the crystals may be dislodged, swept away,and lost. Therefore, the cell pen structure can further be employed incrystallization structures and methods in accordance with the presentinvention to secure crystals in place during the staining process.

FIG. 42 shows an embodiment of a sorting device for crystals based uponthe cell cage concept. Specifically, crystals 8501 of varying sizes maybe formed in flow channel 8502 upstream of sorting device 8500. Sortingdevice 8500 comprises successive rows 8504 of pillars 8506 spaced atdifferent distances. Inlets 8508 of branch channels 8510 are positionedin front of rows 8504. As crystals 8501 flow down channel 8502, theyencounter rows 8504 of pillars 8506. The largest crystals are unable topass between gap Y between pillars 8506 of first row 8504 a, andaccumulate in front of row 8504 a. Smaller sized crystals are gatheredin front of successive rows having successively smaller spacings betweenpillars. Once sorted in the manner described above, the crystals ofvarious sizes can be collected in chambers 8512 by pumping fluid throughbranch channels 8510 utilizing peristaltic pumps 8514 as previouslydescribed. Larger crystals collected by the sorting structure may besubjected to x-ray crystallographic analysis. Smaller crystals collectedby the sorting structure may be utilized as seed crystals in furthercrystallization attempts.

Another factor influencing crystal growth is seeding. Introduction of aseed crystal to the target solution can greatly enhance crystalformation by providing a template to which molecules in solution canalign. Where no seed crystal is available, embodiments of microfluidiccrystallization methods and systems in accordance with the presentinvention may utilize other structures to perform a similar function.

For example, as discussed above, flow channels and chambers ofstructures in accordance with the present invention are typicallydefined by placing an elastomeric layer containing microfabricatedfeatures into contact with an underlying substrate such as glass. Thissubstrate need not be planar, but rather may include projections orrecesses of a size and/or shape calculated to induce crystal formation.In accordance with one embodiment of the present invention, theunderlying substrate could be a mineral matrix exhibiting a regulardesired morphology. Alternatively, the underlying substrate could bepatterned (i.e. by conventional semiconductor lithography techniques) toexhibit a desired morphology or a spectrum of morphologies calculated toinduce crystal formation. The optimal form of such a substrate surfacemorphology could be determined by prior knowledge of the targetcrystals.

Embodiments of crystallization structures and methods in accordance withthe present invention offer a number of advantages over conventionalapproaches. One advantage is that the extremely small volumes(nanoliter/sub-nanoliter) of sample and crystallizing agent permit awide variety of recrystallization conditions to be employed utilizing arelatively small amount of sample.

Another advantage of crystallization structures and methods inaccordance with embodiments of the present invention is that the smallsize of the crystallization chambers allows crystallization attemptsunder hundreds or even thousands of different sets of conditions to beperformed simultaneously. The small volumes of sample and crystallizingagent employed in recrystallization also result in a minimum waste ofvaluable purified target material.

A further advantage of crystallization in accordance with embodiments ofthe present invention is relative simplicity of operation. Specifically,control over flow utilizing parallel actuation requires the presence ofonly a few control lines, with the introduction of sample andcrystallizing agent automatically performed by operation of themicrofabricated device permits very rapid preparation times for a largenumber of samples.

Still another advantage of crystallization systems in accordance withembodiments of the present invention is the ability to control solutionequilibration rates. Crystal growth is often very slow, and no crystalswill be formed if the solution rapidly passes through an optimalconcentration on the way to equilibrium. It may therefore beadvantageous to control the rate of equilibration and thereby promotecrystal growth at intermediate concentrations. In conventionalapproaches to crystallization, slow-paced equilibrium is achieved usingsuch techniques as vapor diffusion, slow dialysis, and very smallphysical interfaces.

However, crystallization in accordance with embodiments of the presentinvention allows for unprecedented control over the rate of solutionequilibrium. In systems metering crystallizing agent by volumeexclusion, the overlying membrane can be repeatedly deformed, with eachdeformation giving rise to the introduction of additional crystallizingagent. In systems that meter crystallizing agent by volume entrapment,the valves separating sample from crystallizing agent may be opened fora short time to allow for partial diffusive mixing, and then closed toallow chamber equilibration at an intermediate concentration. Theprocess is repeated until the final concentration is reached. Either thevolume exclusion or entrapment approaches enables a whole range ofintermediate concentrations to be screened in one experiment utilizing asingle reaction chamber.

The manipulation of solution equilibrium over time also exploitsdifferential rates of diffusion of macromolecules such as proteinsversus much smaller crystallizing agents such as salts. As large proteinmolecules diffuse much more slowly than the salts, rapidly opening andclosing interface valves allows the concentration of crystallizing agentto be significantly changed, while at the same time very little sampleis lost by diffusion into the larger volume of crystallizing agent.Moreover, as described above, many crystallization structures describedreadily allow for introduction of different crystallizing agents atdifferent times to the same reaction chamber. This allows forcrystallization protocols prescribing changed solvent conditions overtime.

6. Experimental Results

In order to compare results of crystallization utilizing the currentprotein crystallization chip with results from two current popularmacroscopic methods of protein crystallization (hanging drop vapordiffusion and microbatch) a number of experiments were performed. Theresults are discussed below.

Model proteins were chosen based on their availability, and difficultyto crystallize. The difficulty of protein crystallization was defined asthe number of conditions claimed by vendor Hampton Research (LagunaNigel, Calif.) to crystallize the protein using vapor diffusion ormicrobatch techniques. Difficulty levels of easy, medium, and difficultare assigned to proteins for which greater than 10 of 48, between 5 and10, and less than 5 conditions will produce crystallization. Both easyand difficult proteins were chosen.

Difficult to crystallize proteins were chosen to apply a stringent testto the chip, to show whether the chip can crystallize proteins requiringa very specific crystallization condition, or even detectcrystallization conditions not detectable using standard crystallizationtechniques. Easy proteins were chosen to better understand thedifferences in behavior between the chip and conventional techniques,and to discover which conditions are incompatible with the chip. Themodel proteins employed in this study were glucose isomerase (medium)proteinase K (easy), beef liver catalase (difficult), bovine pancreastrypsin (medium), lysozyme (easy), and xylanase (medium), and the Bsubunit of topoisomerase VI (medium, not previously crystallized).

Embodiments of the invention present a viable alternative toconventional crystallization methods. Microfluidic devices offerparsimonious use of protein, simpler and more rapid experimental set up,and reduced storage space requirements as compared with conventionalmethods. As discussed below, in nearly all protein models, the chipproduced more hits then conventional methods. It has further been shownthat large high quality crystals may be grown in chip, and that it ispossible to harvest these crystals directly from the chip.

Several routes from the chip to an x-ray beam for collecting areproposed, including harvesting of crystals directly from the chip.Correspondence of about 50–80% between the chip and conventional methodshas been determined, a level of correspondence approximately the same asbetween conventional methods. An analysis of the chip equilibrationsuggests that macro free-interface diffusion might better emulate thechip. This analysis further shows that the chip samples a larger regionin phase space, and hence is more likely to encounter favorablecrystallization conditions.

While permeability of the PDMS material of the chip is a significantfactor in crystallization, nearly all conditions in the Hampton screenproduced crystal hits over the course of target screening. The use of apermeable material in the development of a macroscopic methodcorresponding to chip conditions may be beneficial, as has been shown tobe the case in micro-batch.

a. Reagents and Proteins

Crystal Screen Kit 1, Izit dye, greased Linbro plates, and siliconizedcoverslips were purchased from Hampton Research. Costar 96 well roundbottom plates were purchased from VWR (West Chester, Pa.). HEPES waspurchased from Fluka (St. Louis, Mo.). Calcium chloride, PMSF,benzamidine hydrochloride, and mineral oil were obtained from SigmaAldrich (St. Louis, Mo.).

Glucose isomerase was obtained from Hampton Research and dialyzed intodistilled, deionized water at 4° C. overnight. The protein concentrationwas approximately 30 mg/mL. Protein was aliquotted into 1 mL samples,snap frozen in liquid nitrogen and stored at −20° C.

Proteinase K was obtained from Worthington Biochemicals (Lakewood,N.J.), dialyzed for 5 to 6 hours at 4 C into 1 mM calcium chloride, 25mM HEPES buffer, pH 7.0, filtered through a 0.22 m syringe filter,aliquotted into 100 L samples, and stored frozen at −20° C. Proteinase Kwas approximately 20 mg/mL and the inhibitor PMSF was added prior to usein some experiments at a final concentration of 1 mM.

Beef liver catalase was purchased from Sigma-Aldrich, dialyzed overnightat 4° C. into 25 mM HEPES, pH 7.0, aliquotted into 1 mL samples, snapfrozen in liquid nitrogen, and stored at −20° C. Beef liver catalase wasapproximately 30 mg/mL and centrifuged at 12,000 rpm for 5 minutes priorto use, changing the solution from a dark brown to a slightly tintedsolution.

Lysozyme was purchased from Sigma-Aldrich, and dissolved in 0.2 MolarSodium Acetate (pH 4.7) to a final concentration of 50 mg/mL. Thissolution was then centrifuged in an eppendorf centrifuge (16 000 g) for10 minutes at 4° C.

Xylanase was purchased from Hampton Research. Prior to crystallizationexperiments, the stock solution containing 36 mg/mL protein, 43%Glycerol, and 0.18 Molar Ma/K Phosphate, was diluted by half withdeionized water.

Bovine pancreas trypsin was dialyzed for 5–6 hours at 4° C. into 10 mMcalcium chloride, 25 mM HEPES buffer, pH 7.0, filtered through a 0.22 μmsyringe filter, aliquotted into 100 μL samples, and stored frozen at−20° C. Bovine pancreas trypsin was approximately 60 mg/mL and contained10 mg/mL of the inhibitor benzamidine hydrochloride. One additionalprotein, with an unpublished structure, was also evaluated in chip. TheB subunit of topoisomerase VI is a 50 KDa, ATP-binding, force-generatingsubunit of an archaeal type IIB topoisomerase complex. This protein wasprepared at a concentration of 12 mg/mL in a 100 mMol solution of NaCl,buffered at pH 7.0 with 20 mM TRIS.

Negative controls of buffer without protein were set up on chip to helpdetermine the difference between salt crystals and protein crystals. Onchip no protein controls comprised one chip containing 20 mM calciumchloride, in 25 mM HEPES, buffered at pH 7.0, and one chip containing 20mM calcium chloride, in 1 mM HEPES, buffered at pH 7.0. Individual noprotein controls for Xylanase, Lysozyme, Glucose Isomerase, and the Bsubunit of topoisomerase VI were conducted using the specific buffersdescribed above. Controls were also set up in microbatch with 1 mMcalcium chloride, 25 mM HEPES, pH 7.0, and distilled deionized water.

c. Crystallization Utilizing Hanging Drop

Conventional hanging drop techniques involve hermetically sealing thetarget molecule(s)/crystallization-agent mixture (referred to as the‘drop’) over a well of some type of fluid with a higher osmoticpotential than the drop (typically a higher concentration ofcrystallizing-agent mix) to induce the slow dehydration of the drop witha concomitant increase in concentration of both the target molecule andthe crystallization reagents in the drop. As this concentration processoccurs, the target is slowly driven out of the liquid phase and into asolid phase, hopefully in a crystalline form.

Hanging drop experiments were performed in greased Linbro 24 wellplates. 500 μL of Hampton Crystal Screens 1–48 were placed in the bottomof the well. 1 μL of protein and 1 μL of the crystal screen werecombined in the center of a siliconized glass coverslip. The coverslipwas sealed over the well containing 0.5 mL of screen solution, and theplates were kept at ambient temperatures over a period of two weeks.Plates were monitored for crystal growth daily over a period of one totwo weeks. For duplicates, both drops were placed on a single coverslipover the same reagent well.

d. Crystallization Utilizing Microbatch

Microbatch another conventional crystallization approach. Microbatch issimilar to the hanging drop technique described above, but involvesplacing the ‘drop’ under some type of impermeable or semi-permeablevapor barrier such as oil. Over time, in a manner similar to the vapordiffusion occurring in hanging drop, crystallization reagents promoteaggregation of the target, again, preferably in a crystalline state.

Microbatch experiments were performed in 96 well plates. 100 μL ofmineral oil was pipetted into each well. 1 μL of Hampton Crystal Screens1–48 was added to each well followed by 1 μL of protein and the platewas centrifuged at 1000 rpm for 5 minutes to mix the two drops below theoil layer. The plates were kept at ambient temperatures up to two weeksand monitored daily for crystal growth.

e. On-Chip Crystallization

The particular design for the crystallization chip utilized in theexperiments is shown in FIGS. 45A–C. FIG. 45A shows a simplified planview of the alternative embodiment of the chip. FIG. 45B shows asimplified enlarged plan view of a set of three compound wells. FIG. 45Cshows a simplified cross-sectional view of the wells of FIG. 45B alongline C–C′. This chip design employed metering of target solution andcrystallizing agent utilizing the volume entrapment technique.

Specifically, each chip 9100 contains three compound wells 9102 for eachof the 48 different screen conditions, for a total of 144 assays perchip. A compound well 9102 consists of two adjacent wells 9102 a and9102 b etched in a glass substrate 9104, and in fluidic contact via amicrochannel 9106 In each of the compound wells 9102, the proteinsolution is combined with the screen solution at a ratio that is definedby the relative size of the adjacent wells 9102 a–b. In the particularembodiment shown in FIGS. 45A–C, the three ratios were (protein:solution) 4:1, 1:1, and 1:4. The total volume of each assay, includingscreen solution, is approximately 25 nL. However, the present inventionis not limited to any particular volume or range of volumes. Alternativeembodiments in accordance with the present invention may utilize totalassay volumes of less than 10 nL, less than 5 nL, less than 2.5 nL, lessthan 1.0 nL, and less than 0.5 nL.

The chip control layer 9106 includes an interface control line 9108, acontainment control line 9110 and two safety control lines 9112. Controllines 9108, 9110, and 9112 are filled with water rather than air inorder to maintain a humid environment within the chip and to preventdehydration of the flow channels and chambers in which crystallizationis to be performed.

The interface valves 9114 bisect the compound wells 9102, separating theprotein from the screen until completion of loading. Containment valves9116 block the ports of each compound well 9102, isolating eachcondition for the duration of the experiment. The two safety valves 9118are actuated during protein loading, and prevent spillage of proteinsolution in the event of a failed interface valve.

Fabrication of the microfluidic devices utilized in the experiments wereprepared by standard multilayer soft lithography techniques and sealedto an etched glass slide by baking at 80° C. for 5 hours or greater. Theglass substrate is masked with a 16 um layer of 5740 photoresist, and ispatterned using standard photolithography. The glass substrate is thenetched in a solution of 1:1:1 (BOE:H₂O :2N HCl) for 60 minutes, creatingmicro-wells with a maximum depth of approximately 80 μm.

The chip fabrication protocol just described is only one example of apossible embodiment of the present invention. In accordance withalternative embodiments, the crystallization chambers and flow channelscould be defined between a planar substrate and a pattern of recessesformed entirely in the lower surface of the elastomer portion. Stillfurther alternatively, the crystallization chambers and flow channelscould be defined between a planar, featureless lower surface of theelastomer portion and a pattern of recesses formed entirely in thesubstrate.

Crystallization on chip is set up as follows. All control lines in chipcontrol layer 9106 are loaded with water at a pressure of 15–17 psi.Once the control lines are filled and valves 9114 and 9116 arecompletely actuated, the containment valve 9116 is released, and proteinis loaded through the center via 9120 using about 5–7 psi. The proteinsolution completely fills the protein side of each compound well 9102.Failed valves, if present, are then identified, and vacuum grease isplaced over the corresponding screen via to prevent subsequentpressurization, and possible contamination of the remaining conditions.2.5 to 4 μL of a sparse matrix screen (typically Hampton Crystal ScreenI, 1–48) are then pipetted into the screen vias 9122. The safety valves9118 are released, and a specially designed chip holder (describedbelow) is used to create a pressurized (5–7 psi) seal over all 48 screenvias 9122. The screen solutions are dead end loaded, filling the screenside of each compound well. Protein and crystal screen reagents are keptseparate with the interface valve until all wells are loaded, at whichpoint the containment valve is closed and the interface valve opened toallow diffusion between liquid volumes present in the two halves of thecompound wells 9102

f. Chip Performance

Chip performance varied between experiments. Initially, the chipsexhibited an interface valve failure rate of approximately 30%. However,later experiments typically had between about 3–10 failures out of 48conditions. A total of nine chips had a 100% success rate.

The average protein solution volume used per chip, as measured bytracking the protein meniscus, was approximately 3 μL.

For these experiments, the average time spent setting up an experiment,including filling control lines, was approximately 35 min, with thefastest experiment taking only 20 minutes to set up. This set up timecould potentially be reduced even further through the use of roboticpipetting of solutions to the chip, or through the use of pressures toload and prime delivered solutions, as discussed below in conjunctionwith FIG. 80.

g. General Crystal Growth Observation and Data Analysis

Hanging drop, microbatch, and chip experiments were observed for hitsfor up to 2 weeks. Unless otherwise specified, hits are defined assingle crystals, needle crystals, plate crystals, rod crystals,spherulites, or precipitate. Phase separation or oil droplets are notcounted as hits. In some cases, crystals were confirmed to be proteincrystals by dying with Izit dye, which does not stain salt crystals.Izit dye was diluted 1/20 in the corresponding crystal screen and 1 μLwas added to the crystal drop. Protein crystals were also confirmed byprobing the crystal with a crystal probe.

Comparisons on crystal growth patterns between techniques were based onsimilarities in conditions producing hits. For duplicate and triplicatetests, comparisons were made such that two techniques were considered tohave similar growth patterns if one or both of the duplicates produced ahit in both techniques (e.g. chip and microbatch are considered to havesimilar crystal growth patterns if chip 1 or chip 2 produced a hit andmicrobatch 1 or microbatch 2 produced a hit.).

h. Glucose Isomerase Crystallization

Glucose isomerase crystallization was set up in one hanging dropexperiment, two microbatch experiments, and on six separate chips.Hanging drop experiments were monitored daily for one week, and thenevery other day for a second week, microbatch plates were observed fortwo weeks, and chips were monitored for up to 9 days. The results wereconsistent between chips when other factors (valve failure, failure toload with protein or crystal screen) were taken into consideration.

Results of these experiments are summarized in the Venn diagram shown inFIG. 46. One crystal screen (reagent #33) failed to produce crystals orprecipitate in any of the experiments. 33 of the 48 screens producedhits in the hanging drop experiments; 28 of 48 produced hits in themicrobatch method, and 47 of 48 produced hits on chip. There wassignificant agreement between the microbatch and hanging dropexperiments: 81% of the microbatch results agreed with hanging dropresults, where 13 conditions produced no hits in either of thetechniques, and 26 conditions produced crystal or precipitate in bothtechniques.

There was less agreement between the chip and microbatch results (60%agreement) or between the chip and hanging drop methods (71%). Twelveconditions that were observed to produce hits in the chip, which did notin the other methods. Table 1 shows the results of glucose isomerasecrystallization in the three techniques.

TABLE 1 Glucose Isomerase Crystallization Results micro- hanging Reagentprecipitant salt chip batch drop number classification conditionacid/base day 9 day 14 day 14 1 organic low acid C1 C1 C1 2 salt high nobuffer C1 3 salt high no buffer C1 4 salt high base C1 C1 5 organic highweak base C1 C1 6 polymer high base C1 C1 C1 7 salt high weak acid A1 8organic high weak acid C1 C1 9 polymer high acid A1 A1 C1 10 polymerhigh acid C1 A1 C1 11 salt high acid C1 A1 12 organic high weak base C1A1 A1 13 polymer high base C1 14 polymer high weak base A1 A1 A1 15polymer high weak acid A1 A1 A1 16 salt high weak base C1 17 polymerhigh base C1 A1 C1 18 polymer high weak acid B1 B1 C1 19 organic highbase A1 20 polymer high acid A1 C1 C1 21 organic high weak acid C1 A1 A122 polymer high base A1 A1 A1 23 polymer high weak base B1 A1 A1 24organic high acid A1 B1 C1 25 salt high weak acid B1 26 organic highacid B1 27 organic high weak base A1 28 polymer high weak acid C1 C1 C129 salt high weak base B1 30 polymer high no buffer C1 C1 C1 31 polymerhigh no buffer C1 A1 C1 32 salt high no buffer C1 A1 C1 33 salt high nobuffer 34 salt high acid C1 35 salt high weak base C1 A1 C1 36 polymernone base C1 C1 37 polymer none acid B1 C1 38 salt high weak base A1 C139 polymer none weak base C1 C1 40 organic none acid B1 A1 A1 41 organicnone weak base B1 42 polymer low no buffer C1 C1 C1 43 polymer none nobuffer C1 C1 44 salt high no buffer C1 A1 A1 45 polymer high weak acidC1 C1 C1 46 polymer high weak acid B1 B1 C1 47 salt high acid C1 C1 C148 salt high base C1 C1 C1 X49 polymer high no buffer X X X X50 polymerhigh no buffer X X X 47 28 33 Key: A1 = Crystals; B1 = Needle; Plate; orRod Microcrystals; C1 = Precipitate; X = N/A or Failed Valve

Where crystal growth was observed in microbatch and hanging dropmethods, the crystals were confirmed to be proteins by poking thecrystal with a crystal probe. As shown in FIGS. 47A–B crystals thatcrumble under pressure (FIG. 47B) should be protein. Crystals that donot crumble or shatter (FIG. 47A) are salt crystals. As shown in FIGS.48A–B large high quality crystals were produced utilizing the chip.

Glucose Isomerase crystallization on chip vs. microbatch was alsoevaluated in a separate set of experiments. The Glucose Isomerase wasdialyzed against deionized water to a final concentration of 31 mg/mL.Hits in these experiments were defined as crystals, microcrystals,needles, plates, rods, or spherulites, while precipitation was notcounted as a hit. The microbatch experiment was run for 2 weeks, whilethe chip experiment was run for 3 days. At the end of three days, thechip became dehydrated due to insufficient water in the containmentcontrol tube. The results of these experiments are summarized in theVenn Diagram shown in FIG. 49.

The identity of the crystals, and the reproducibility of the results,was investigated in the following experiment. The hits from the initialscreen, Hampton conditions 3, 4, 6, 9, 10, 14, 15 (in triplicate), 17,18, 20, 22, 28, 30, 32, 38, 39, 42, 43, and 46 (in duplicate), were setup again on a single chip. On the same chip, 2 protein vs. watercontrols, and a complete set of water vs. screen controls were set up.All of the 22 conditions again gave hits. Both protein vs. water, andall 24 water vs. screen controls were clear (except for some phaseseparation). The duplicate condition 46 wells showed similar crystals inthe mpms (medium protein: medium solution) and spls (small protein:large solution) wells, with morphology depending on the protein tosolution ratio. The lpss (large protein: small solution) well was clearin both cases. The triplicate condition 15 showed crystals in all mpmsand spls conditions, and was clear in the lpss condition. The morphologyof all the spls conditions was identical, while one of the mpmsconditions showed a different morphology.

A comparison of the three condition 15 results is shown in FIG. 50. Manyof the crystals were large, and of x-ray diffraction quality, showinglargest dimensions greater than 150 um, and smallest dimension ofapproximately 30 um. The first crystals appeared after 4 hrs ofincubation, and approximately 80% of crystals had appeared after 1 day.A gallery of these pictures is shown in FIG. 51. All crystals are shownafter a single day of incubation.

i. Proteinase K

Proteinase K was crystallized in two microbatch, two hanging drop, andthree microfluidie chip experiments. The two microbatch experiments andthe two hanging drop experiments were monitored for seven days. Threechip experiments were monitored, one for five days, another for sixdays, and a third for seven days. Due to the difficulty of crystallizingproteinase K, two conditions, proteinase K with the inhibitor PMSF andproteinase K without the inhibitor PMSF, were employed.

FIG. 52 is a Venn diagram summarizing the crystallization results on thelast day of observation. The results show that protein crystallizationof proteinase K is relatively inconsistent from technique to technique.Out of 48 conditions, only 15 conditions, or 31%, gave similar resultsacross all three techniques. There were 5 conditions that produced hitsin all three techniques and 10 conditions in which no crystals orprecipitate was found. Eight conditions produced hits in the chip only,10 conditions produced hits in microbatch only, and 4 conditionsproduced hits in hanging drop only. Only 18 out of 38 hits or no hits(28 total hits in chip and hanging drop and 10 no hits in either method)were common to the chip and hanging drop (47%). Twenty out of 44 hits orno hits were common to the chip and microbatch (45%). Eighteen out of 40hits or no hits were in common to the hanging drop and microbatch (45%).Table 2 shows the actual results of proteinase K crystallization in thethree techniques.

TABLE 2 Proteinase K Crystallization Results. microbatch microbatchHanging hanging chip 3 Rea- 1 w/o 2 w/ drop 1 drop 2 chip 1 chip 2 w/ogent precipitant salt PMSF PMSF w/PMSF w/PMSF w/PMSF w/PMSF PMSF numberclassification condition acid/base day 7 day 7 day 10 day 10 day 7 day 6day 5 1 organic low acid A1 X A2 salt high no buffer A1 X A3 salt highno buffer X X A4 salt high base C1 C1 X 5 organic high weak base X X A6polymer high base A1 X A7 salt high weak acid C1 8 organic high weakacid C1 X A9 polymer high acid C1 C1 C1 A10 polymer high acid A1 A1 A11salt high acid A1 12 organic high weak base A1 B1 13 polymer high base X14 polymer high weak base X A15 polymer high weak acid C1 B1 X A16 salthigh weak base C1 C1 X A17 polymer high base B1 A1 X C1 X A18 polymerhigh weak acid A1 C1 19 organic high base A1 A20 polymer high acid A1 C1B1 21 organic high weak acid A22 polymer high base A1 23 polymer highweak base 24 organic high acid X A25 salt high weak acid A1 26 organichigh acid C1 27 organic high weak base A28 polymer high weak acid A1 A29salt high weak base A1 A30 polymer high no buffer B1 A1 C1 C1 C1 C1 B1A31 polymer high no buffer A1 C1 C1 C1 B1 A32 salt high no buffer C1 C1B1 A33 salt high no buffer C1 C1 A34 salt high acid A1 C1 A35 salt highweak base A1 C1 C1 X A36 polymer none base A1 X 37 polymer none acid A38salt high weak base C1 C1 C1 C1 X A39 polymer none weak base A1 B1 A40organic none acid A1 C1 C1 C1 C1 C1 A41 organic none weak base A1 A42polymer low no buffer A1 A1 C1 C1 43 polymer none no buffer A1 C1 A44salt high no buffer A45 polymer high weak acid X C1 C1 A46 polymer highweak acid C1 A47 salt high acid B1 C1 C1 C1 B1 A48 salt high base A1 C1C1 X 49 polymer high no buffer X X X X X X X 50 polymer high no buffer XX X X X X X 11 13 16 12 10 12 12 Key: A1 = Crystal; B1 = Needle; Plate;or Rod; C1 = Precipitate; X = N/A or Failed Valve

These results indicate that crystallization is a stochastic event,depending on technique, nucleation, and other variables. Hits were seenin some techniques, but not others. Hits were also seen in oneduplicate, but not the other. Where duplicates in hanging drop were setup in the same well on the same coverslip, hits were even seen in onedrop, but not a neighboring drop, not an atypical result forcrystallization of certain proteins. Also, although there are manyoverlaps on hits between techniques, the types of hits vary. Many hitsproduced by microbatch are single crystals, some hits produced byhanging drop are crystals and most are precipitate, and hits produced inthe chip tend to be either needles or precipitate.

FIG. 53A shows proteinase K crystal observed in microbatch. FIG. 53Ashows needles observed on chip. Reagent 30 is a good example of thevariations on the types of hits. In microbatch, a crystal formed in oneduplicate and needles were produced in the other, precipitate was foundin hanging drop, and one chip resulted in precipitate, while the otherresulted in needle crystals.

The chip was able to produce hits in conditions that contained acidicbuffers (pH 5.6 or pH 4.6), basic buffers (pH 8.5), and even isopropylalcohol. Furthermore, of the 17 conditions that produced hits in hangingdrop or microbatch, but not in the chip, the reagents ranged fromorganic or polymer precipitant to salt precipitant, from high salt to nosalt, and from acidic buffers to basic buffers. These results indicatethat the chip can withstand acids, bases, and organic materials used inthe Hampton crystallization screens.

The PDMS elastomer material utilized in certain embodiments of thepresent invention is generally compatible with most solvents used inprotein crystallization. A nonexclusive list of solvents that are notcompatible with PDMS includes concentrated acids such as hydrofluoricacid, nitric acid, sulfuric acid, and Aqua Regia, as well asbenzaldehyde, benzene, carbon tetrachloride, chlorobenzene, chloroform,cyclohexane, ether, diethyl ether, isopopyl ether, methyl ketone, ethelketone, methylene chloride, petroleum ether, tetrahydrofuran, toluene,trichloroethylene, acetates, and xylene. Where such materials are to beutilized in conjunction with PDMS-based microfluidic devices inaccordance with embodiments of the present invention, a surface coatingor modification of the PDMS may be required. Alternatively, PDMS may bereplaced by a different elastomer material having appropriate solventcompatibility. A large number of possible alternative elastomermaterials have been discussed previously.

j. Beef Liver Catalase Crystallization

Beef liver catalase was crystallized in two chips, two hanging drops,and one microbatch experiment. Crystal screens 1–48, excluding 6, weretested in duplicate in hanging drop and carried out for 7 days. Crystalscreens 25–36 were tested in duplicate in the chip for 3 days andcrystal screens 1–48 were set up in another chip and monitored for 7days. Crystal screens 1–48 in microbatch were observed for 7 days. Asshown in FIG. 54, in some cases crystals grew in wells experiencingsignificant dehydration. Table 3 shows results of the crystallizationfor beef liver catalase.

TABLE 3 Beef Liver Catalase Crystallization Results Rea- chip 1 chip 1hanging drop hanging drop gent precipitant salt top bottom chip 2 1 2microbatch number classification condition acid/base day 3 day 3 day 7day 7 day 7 day 7  1 organic low acid X X C1 C1 C1  2 salt high nobuffer X X A1  3 salt high no buffer X X C1 C1 C1 C1  4 salt high base XX C1 C1 C1  5 organic high weak X X A1 C1 C1 base  6 polymer high base XX B1 X X C1  7 salt high weak X X A1 acid  8 organic high weak X X C1 C1C1 acid  9 polymer high acid X X B1 C1 C1 B1 10 polymer high acid X X B1C1 C1 C1 11 salt high acid X X C1 C1 C1 12 organic high weak X X C1 C1base 13 polymer high base X X A1 14 polymer high weak X X C1 base 15polymer high weak X X B1 C1 C1 B1 acid 16 salt high weak X X C1 C1 C1 C1base 17 polymer high base X X B1 C1 C1 C1 18 polymer high weak X X B1 C1C1 B1 acid 19 organic high base X X A1 C1 C1 20 polymer high acid X X B1C1 C1 C1 21 organic high weak X X A1 acid 22 polymer high base X X B1 C1C1 C1 23 polymer high weak X X base 24 organic high acid X X C1 C1 C1 C1 25* salt high weak C1 A1 C1 acid 26 organic high acid A1 A1 A1 C1 C1 27organic high weak A1 A1 C1 C1 base 28 polymer high weak B1 C1 B1 C1 C1B1 acid 29 salt high weak C1 C1 C1 B1 C1 base 30 polymer high no bufferB1 C1 B1 C1 C1 C1 31 polymer high no buffer B1 B1 B1 C1 C1 B1 32 salthigh no buffer A1 C1 C1 C1 B1 33 salt high no buffer C1 C1 C1 C1 C1 C134 salt high acid C1 C1 C1 C1 C1 C1 35 salt high weak C1 C1 C1 base  36*polymer none base B1 B1 B1 C1 B1 37 polymer none acid X X B1 C1 C1 C1 38salt high weak X X C1 C1 A1 C1 base 39 polymer none weak X X C1 C1 C1 B1base 40 organic none acid X X B1 C1 C1 B1 41 organic none weak X X B1 C1C1 B1 base 42 polymer low no buffer X X C1 C1 C1 C1 43 polymer none nobuffer X X B1 C1 C1 44 salt high no buffer X X 45 polymer high weak X XC1 C1 acid 46 polymer high weak X X B1 C1 C1 B1 acid 47 salt high acid XX C1 C1 C1 C1 48 salt high base X X C1 C1 X49   polymer high no buffer XX X X X X X50   polymer high no buffer X X X X X X sum 25– 12 11 11 1010 6 36 sum 1–48 12 11 43 36 36 28 Key: A1 = Crystal; B1 = Needle;Plate; or Rod; C1 = Precipitate; X = N/A or Failed Valve; * =Dehydration

Beef liver catalase shows more promising conditions in the chip than inconventional hanging drop techniques. As shown in the Venn diagram ofFIG. 55 conditions produced hits in the chip versus 36 in hanging dropand 28 in microbatch. Only 2 conditions resulted in no hits in any ofthe three techniques. This was contrary to expectations that beef livercatalase is a difficult protein to crystallize, as Hampton researchreported crystals in only 2 out of 48 screens.

Initially it was hypothesized that the chip might mimic the microbatchcondition over hanging drop. However, the chip produced results moresimilar to hanging drop than microbatch. Thirty-six out of 48 hits or nohits were common to the chip and hanging drop (75%). Twenty-nine out of47 (62%), and 27 out of 41 (66%) were in common between the chip andmicrobatch, and between microbatch and hanging drop, spectively.

FIG. 56A shows that beef liver catalase crystals obtained byconventional microbatch techniques resulted in a morphology similar tocrystals formed on chip (FIG. 56B). Eight out of 17 hits that wereneedles in the chip were also needles in microbatch.

k. Bovine Pancreas Trypsin Crystallization

Bovine Pancreas Trypsin was set up once each in hanging drop,microbatch, and chip. Conditions 1–24 were set up on one chip, andconditions 25–48 were set up on a second chip. The chip containingconditions 25–48 lost containment on day 4, but no change or few changesare expected in those conditions after day 4. Microbatch and hangingdrop experiments were observed for 7 days and microbatch crystals wereconfirmed, similar to glucose isomerase, with a crystal probe. Table 4summarizes the data from the experiment.

TABLE 4 Bovine Pancreas Trypsin Crystallization Results. micro- hangingReagent precipitant salt batch drop chip number classification conditionacid/base day 7 day 7 day 6 1 organic low acid 2 salt high no buffer 3salt high no buffer A4 salt high base C1 C1 5 organic high weak base 6polymer high base 7 salt high weak acid 8 organic high weak acid 9polymer high acid C1 10 polymer high acid 11 salt high acid 12 organichigh weak base 13 polymer high base 14 polymer high weak base A15polymer high weak acid A1 A1 C1 A16 salt high weak base C1 17 polymerhigh base A1 B1 18 polymer high weak acid C1 19 organic high base C1 A20polymer high acid C1 21 organic high weak acid 22 polymer high base 23polymer high weak base 24 organic high acid 25 salt high weak acid 26organic high acid C1 27 organic high weak base X A28 polymer high weakacid 29 salt high weak base A30 polymer high no buffer A1 A1 A1 A31polymer high no buffer A1 C1 A1 32 salt high no buffer C1 33 salt highno buffer C1 34 salt high acid 35 salt high weak base 36 polymer nonebase 37 polymer none acid 38 salt high weak base C1 39 polymer none weakbase C1 40 organic none acid C1 41 organic none weak base 42 polymer lowno buffer C1 43 polymer none no buffer 44 salt high no buffer 45 polymerhigh weak acid C1 46 polymer high weak acid 47 salt high acid C1 48 salthigh base X49 polymer high no buffer X X X X50 polymer high no buffer XX X 4 11 12 Key: A1 = Crystal; B1 = Needle; Plate, or Rod; C1 =Precipitate; X = N/A or Failed

As seen in Table 4, 28 conditions did not give hits in any of the threemethods and only 3 conditions gave hits in all three techniques. Sevenconditions resulted in hits in the chip only, and 7 other conditionsproduced hits in hanging drop only. No conditions were unique tomicrobatch. Despite the unique hits in chip and hanging drop, there isgood agreement between the three methods. Thirty-two out of 48conditions produced hits both in chip and hanging drop (67%), 32 out of41 conditions resulted in hits in chip and microbatch (78%), and 31 outof 41 conditions gave hits in microbatch and hanging drop (76%).

FIG. 58A shows Bovine pancreas trypsin crystals observed in microbatch.FIG. 58B shows Bovine pancreas trypsin crystals observed from the chip.

l. Lysozyme Crystallization

The crystallization of Lysozyme was evaluated, in chip, microbatch, andin hanging drop. Hits in these experiments were defined as crystals,microcrystals, needles, plates, rods, or spherulites, whileprecipitation was not counted as a hit. The microbatch experiment wasrun for 2 weeks, while the chip experiment was run for 7 days. Thehanging drop experiment was terminated after 3 days due to temperaturefluctuations that resulted from the plates being left in directsunlight. It is likely that had the hanging drop experiment beencontinued, further crystallization conditions would have been revealed.The results of these experiments are summarized in the Venn Diagramshown in FIG. 59.

Inspection of FIG. 59 shows that the chip revealed the mostcrystallization conditions, and that approximately 40% of thesecrystallization conditions were also observed using conventionaltechniques. This agreement may have been improved had the hanging dropexperiments not been terminated prematurely. Also of note is thatalthough Lysosyme is typically considered a very easy protein tocrystallize, and gave numerous hits both in chip, and in hanging drop,it showed only 4 hits in the micro batch experiment.

Contrary to the results for Glucose Isomerase, in which crystallizationwas accelerated in chip, crystallization of Lysosyme was slower in chip.While one day revealed 63% of hits in hanging drop, and 75% of hit inmicro-batch, only 1 of 23 chip hits occurred within the first 24 hrs ofincubation.

All three methods produced large single crystals of x-ray diffractionquality. FIGS. 60A–B show a comparison of crystals formed in chip andmicrobatch, respectively.

m. Xylanase Crystallization

The crystallization of Xylanase was evaluated, in chip, microbatch, andin hanging drop. Hits in these experiments were defined as crystals,microcrystals, needles, plates, rods, or spherulites, whileprecipitation was not counted as a hit. The microbatch experiment wasrun for 2 weeks, while the chip experiment was run for 10 days. Thehanging drop experiment was terminated after 3 days due to temperaturefluctuations that resulted from the plates being left in directsunlight. It is likely that had the hanging drop experiment beencontinued, further crystallization conditions would have been revealed.The results of these experiments are summarized in the Venn Diagramshown in FIG. 61.

Inspection of FIG. 61 shows that 75% of the hits in chip were reproducedin hanging drop or micro batch. The highest quality crystals, both inchip, and in microbatch, were formed using condition 14. FIGS. 62A–Bshows a comparison of these crystals, grown in microbatch, and on chip,respectively. While 6 of the 7 micro batch hits occurred within thefirst day of incubation, 7 of the 8 chip hits occurred at incubationtimes greater than 48 hrs. Condition 14 was reproduced with 6-foldredundancy in a subsequent experiment, and showed only two hits(occurring at 72 hrs) during a 7 day incubation. These crystals grew tofull size (approx. 100 μm) over-night. The long time for crystalformation, inconsistent results, and quick subsequent growth of thecrystals, suggests that nucleation is the rate limiting step forcrystallization of Xylanase in chip.

The information provided by Hampton Research indicates that Xylanase canbe crystallized using Na/K Phosphate solution in a pH range of 7 to 8.2,and at concentrations between 0.6 and 1.4 Mol. In two separateexperiments, a systematic screen of Xylanase vs. Na/K Phosphate wasconducted in chip.

One experiment tested 50% stock Xylanase against a grid of Na/KPhosphate conditions, covering pH values from 6.4 to 7.8 in steps of0.2, and concentration from 1.0 Molar to 3.0 Molar in steps of 0.4. Theother experiment tested 25% stock Xylanase against the same grid. After1 day, microcrystals (less than 10 um largest dimension) were seen inboth experiments for all pH values with concentrations higher than 1.4Molar. The wells showed no further change for the next 6 days.

On the seventh day, large plate/star structures where observed in thesame condition (pH 6.4, 1M) on both chips. The 50% Xylanase chip showedthis hit in the mpms (medium protein: medium solvent) condition, whilethe 25% Xylanase chip showed the hit in the lpss (large protein: smallsolvent) condition; consistent with the concentration of protein beingsimilar. These hits are shown in FIGS. 63A–B. This result indicatescrystallization of Xylanase to be sensitive to changes in pH and changesin Na/K Phosphate concentration. The chip/chip correspondence also showsrepeatability to be good, even at long incubation times. Although thehit did occur at the corner of the grid, the result further suggeststhat even after 7 days, conditions on the chip were not sufficientlyblended (in pH and salt concentration), so as to make nearby wells showcrystallization.

n. B Subunit of Topoisomerase VI

The B subunit of topoisomerase VI was tested in chip against thefollowing 3 commercial sparse matrix screens, Hampton Crystal Screen I(HCS1), Hampton Crystal Screen II (HCS2), and Emerald Wizard Screen II(WIZ2). Since only a small volume of protein solution was available (50uL), bulk controls were not done. All three experiments were incubatedfor 8 days. The results of these experiments are tabulated in Table 5.

TABLE 5 B Subunit of Topoisomerase VI Results Morphology HCS1 HCS2 WIZ2Crystals Plates 22, 9, 6, 17, 18, 36, 46, 48 3, 18, 28 37, 28, 41,Needles 15, 40 8, 18 Stars 15, 40, 1, 10 16, 14, 25 7, 8, 24, 47 Micro4, 3, 30, 32, 35, 45, 31, 10, 24, 23, 31, 20, 30, 32, 44, Crystals/ 4336 36 Specs Aggregate 2, 5, 7, 8, 16, 21, 15, 26, 16, 15, 35, 48, 27,29, 33, 25, 37, 38, 47 45, 31, 37 Lost 26, 27 Condition

The chip shows many large plate crystals, with some of sufficient sizefor x-ray diffraction. Two examples of these plates are shown in FIGS.64A–B. Prior experiments have revealed that the B subunit oftopoisomerase VI crystallizes well in PEG conditions, and typicallydisplays the plate morphology. The chip results demonstrate an excellentreproduction of this behavior, with all 14 plate hits being on PEGconditions. Furthermore, the chip outperformed the hanging drop andmicro batch experiments, showing more frequent hits. Comparing the chipresults to those of micro-batch shows that the chip uncovered 23conditions that gave rise to plate crystals, while only 10 werediscovered in micro-batch. Furthermore, 6 of the 10 conditions that gavecrystals in micro-batch also gave crystals in chip, while 3 gavegranular precipitate, and only one did not produce a hit in chip.Lastly, crystals formed in micro-batch were not present for severaldays, while chip crystals began to form in less than 6 hours.

o. Negative Controls

No protein controls were set up on chips to help identify proteincrystals versus salt crystals. Chips were set up as described forprotein crystallization experiments. 1 mM calcium chloride, 25 mM HEPES,pH 7.0 was used as a negative control for proteinase K and 20 mM calciumchloride, 25 mM HEPES, pH 7.0 was used as a negative control for bovinepancreas trypsin (calcium chloride concentration should have been 10 mMand benzamidine hydrochloride was not added). Controls were also set upin microbatch and hanging drop with 1 mM calcium chloride, 25 mM HEPES,pH 7.0, and filtered distilled deionized water. Specific no-proteincontrols (respective buffer with no-protein) were done for Lysosyme,Glucose Isomerase, Xylanase, and the B subunit of topoisomerase VI.Where crystals have been reported, the no-protein controls were clear.

7. Analysis of Crystal Structure from Protein on Chip

The utility of the chip is ultimately dependent on its' ability toquickly generate high quality diffraction patterns at a reduced cost. Aclear path from the chip-to-protein structure is therefore invaluable.Several paths from in-chip crystals to diffraction data are discussedbelow.

a. On-chip Crystallization as Screening for Reproducing Crystals UsingConventional Techniques

One possible application for a chip is determination of favorablecrystallization conditions that can subsequently be reproduced usingconventional techniques. Correspondence between the chip and twoconventional techniques (micro batch and hanging drop) has been shown tobe variable (between 45% and 80%). However, this variation is not afeature unique to the chip. These widely used crystallization techniquesshow only marginal correspondence (e.g. 14 of 16 hanging drop hits forlysosyme do not occur in microbatch), and often show variation withinthemselves, for example Table 2 (Proteinase K). As a tool for screeninginitial crystallization conditions, the chip may be able to identify asmany promising conditions.

FIG. 65 shows a comparison of the number of hits generated on eachprotein using the three different technologies. In FIG. 65 onlycrystals, microcrystals, rods, and needles are counted as hits, whilespherulites and precipitation is not counted. The data on Proteinase Kis a sum of the experiments with and without PMSF, and data for the Bsubunit of topoisomerase VI has not been included for lack of hangingdrop data (although the chip far outperformed micro-batch in this case).Inspection of FIG. 65 shows that in 4 of the 6 cases, the chip producedmore hits than either conventional method. Only for Proteinase K did theconventional techniques provide significantly more favorable results.

In order to understand differences between crystallization methods toidentify possible reasons for productivity of the chip, we mustappreciate that the three methods produce different thermodynamicconditions on both short and long time scales. In order to induceprotein crystallization, one must make the crystallization energeticallyfavorable (supersaturation condition), and maintain these conditionslong enough for crystal growth to occur.

There are also different degrees of supersaturation. In lowsupersaturation, crystal growth tends to be supported, while nucleationof new crystals is relatively unlikely. In high supersaturation,nucleation is rapid, and many small low quality crystals may often beformed. In the three methods considered here, the condition ofsupersaturation is achieved through the manipulation of the relative,and absolute, concentration of protein and counter-solvent.

A comparison of the phase space evolution/equilibration of the threemethods is shown in FIG. 66. For the micro batch technique, mixing ofthe two solutions is quick, and when kept under impermeable oil layers,little significant concentration occurs over time. Micro batch thereforetends to sample only a single point in phase space, maintainingapproximately the same condition over time.

Hanging drop starts out like micro batch, with rapid mixing of the twosolutions, but then undergoes a concentration on a longer timescale(hours to days) due to vapor equilibration with the more concentratedsalt/precipitant reservoir. During the evaporative dehydration of thedrop, the ratio of protein to precipitant remains constant.

As described in detail below in the description of Microfluidic FreeInterfaces, on the short time scale the chip dynamics most closelyresemble a free interface diffusion experiment. Mixing is slow, and therate of species equilibration (protein/precipitant/proton/salt) dependson species' diffusion constants. Small molecules such as salts havelarge diffusion constants, and hence equilibrate quickly. Largemolecules (e.g. proteins) have small diffusion constants, andequilibrate more slowly.

FIGS. 67A–D show photographs of time-resolved equilibration of two dyesin three compound wells. The dye used has a molecular weight ofapproximately 700 Da. From these pictures the equilibration time of thedye is estimated to be approximately 1.5 hours. The dimensional Einsteinequation may be used to get a rough estimate of diffusion times.

$\begin{matrix}\begin{matrix}{{t = \frac{x^{2}}{4D}};\text{where:}} & {{t = {{diffusion}\mspace{14mu}{time}}};} \\\; & {{x = {{longest}\mspace{14mu}{diffusion}\mspace{14mu}{length}}};{and}} \\\; & {D = {{diffusion}\mspace{14mu}{coefficient}}}\end{matrix} & (2)\end{matrix}$Generally, the diffusion coefficient varies inversely with the radius ofgyration, and therefore as one over the cube root of molecular weight.

$\begin{matrix}\begin{matrix}{{D \propto \frac{1}{r} \propto \frac{1}{m^{1/3}}};\text{where:}} & {D = {{diffusion}\mspace{14mu}{coefficient}}} \\\; & {{r = {{radius}\mspace{14mu}{of}\mspace{14mu}{gyration}}};{and}} \\\; & {m = {{molecular}\mspace{14mu}{weight}}}\end{matrix} & (3)\end{matrix}$As compared with the rough 1.5 hr equilibration time for the dye, anapproximate equilibration time for a protein of 20 KDa over the samedistance is estimated to be approximately 45 hours. The equilibrationtime for a small salt of a molecular weight of 100 Da over the samedistance is about 45 minutes.

Soon after the chip interface valves are opened, the proteinconcentration on the protein-side has changed very little, while thesalt concentration has increased to one of three final values determinedby the relative size of the protein and solvent wells. Next, over a timeof approximately 45 hours, the protein concentration equilibrates,increasing on the solvent side, and decreasing on the protein side. Thefinal protein concentration is once again determined by the relativechamber sizes. A similar process occurs on the solvent side of the chip,with solvent concentration starting at its' highest value, and proteinconcentration starting at zero. The chip therefore samples more ofphase-space, and consequently has a better chance of detectingconditions favorable to crystallization. On time scales larger thanthose shown in FIG. 66, there may be subsequent changes in the chipconditions due to the permeability of PDMS to some solutes and solvents.

If it is desirable to slow down or halt the equilibration process, theinterface valves may be gated, adjusting the equilibration rate bychanging the valve actuation duty cycle. This provides the opportunityto gain temporal control of the equilibration process, a key advantageof embodiments in accordance with the present invention. Additionaldiscussion of temporal control over the equilibrium process is describedbelow in conjunction with FIGS. 76A–C.

The crystallization technique of free interface diffusion in capillariesmay more closely emulate the chip results. Traditionally this method isnot often used due to the difficulty of reliably setting up awell-defined interface. However, in microfluidic environments it isrelatively easy to establish reliable free-interface diffusionexperiments. Additional discussion of the formation of microfluidicfree-interfaces is presented below in conjunction with FIGS. 71–75.

It may also be possible to develop a macroscopic crystallizationtechnique, based on free-interface diffusion, emulating chip conditionswhile providing easy harvesting of crystals.

b. On-Chip Crystallization to Obtain Seed Crystals for CrystallizationUsing Conventional Techniques

In another application of the crystallization chip, crystals may begrown for harvesting using conventional methods. Inspection of FIG. 66suggests that exporting a favorable condition from the chip to anothermethod, will likely require screening around this condition. In apreliminary experiment, 10 conditions that produced Glucose Isomerasecrystals in chip, but that did not produce crystals in micro batch, wereused as a basis for additional screening in micro batch. Each conditionwas mixed with the stock protein solution at 1:4, 1:1, and 4:1 ratios,both under paraffin oil, and silicone oil. Silicone oil was used sinceit has permeability similar to that of the PDMS. At the end of 6 days, 6of the ten proteins that did not previously crystallize in micro batchshowed crystals. Of particular interest was that of HCSI-6, which formedhigh quality crystals at a 4:1 ratio of protein to solution, undersilicone oil. This condition showed no crystals in any of the otherconditions, including the identical condition under paraffin oil. FIG.68A shows crystals formed under the silicone oil, and FIG. 68B shows thenegative result under paraffin oil. This result shows that thepermeability of the containment material can have significant impact onprotein crystallization.

This also suggests that materials with permeability similar to that ofPDMS may be useful in establishing a macro crystallization techniquethat more closely resembles the chip.

c. Direct Analysis of Crystals Grown On-Chip

If high quality crystals can be grown in, and extracted from the chip,crystallization conditions need not be exported. As shown in FIGS. 50–51(glucose isomerase), 60A (lysozyme), 62B (Xylanase), and 64A–B (Bsubunit of topoisomerase VI), high quality crystals of sufficient sizefor x-ray crystallography, can be grown on the chip. Since the chip canbe removed from the glass substrate, it is also possible to extractprotein crystals. Six crystals (1 of Xylanase, 5 of the B subunit oftopoisomerase VI) have been removed from chip, mounted in a cryo-loop,and flash frozen in liquid nitrogen. In one case, with a Xylanasecrystal grown in HCS1-14, the chip was peeled off, and 5 uL of 30%glycerol, 70% solvent, was pipetted onto the well. The crystal was thenextracted using a 300 um cryo-loop, and flash-frozen in liquid nitrogen.FIG. 69 shows this Xylanase (the same as shown in FIGS. 62A–B), mountedin the cryo-loop.

d. Growing/Harvesting Chip

As previously described, once a protein crystal has been formed,information about its three dimensional structure can be obtained fromdiffraction of x-rays by the crystal. However, application of highlyenergetic radiation to the protein tends to generate creates heat.X-rays are also ionizing, and can result in the production of freeradicals and broken covalent bonds. Either heat or ionization maydestroy or degrade the ability of a crystal to diffract incident x-rays.

Accordingly, upon formation of a crystal a cryogenic material istypically added to preserve the crystalline material in its alteredstate. However, the sudden addition of cryogen can also damage acrystal. Therefore, it would be advantageous for an embodiment of acrystallization chip in accordance with the present invention to enablethe direct addition of cryogen to the crystallization chamber once acrystalline material is formed therein.

In addition, protein crystals are extremely delicate, and can quicklycrumble or collapse in response to physical trauma. Accordingly,harvesting a crystal unharmed from the small chambers of a chip poses apotential obstacle to obtaining valuable information about thecrystalline material.

Therefore, it would also be advantageous for an alternative embodimentof a crystallization chip in accordance with the present invention toallow direct interrogation by x-ray radiation of crystalline materialsformed in a chip, thereby obviating entirely the need for separatecrystal harvesting procedures.

Accordingly, FIG. 71A shows a plan view of a simplified embodiment of acrystal growing chip in accordance with the present invention. FIG. 71Bshows a simplified cross-sectional view of the embodiment of the crystalgrowing chip shown in FIG. 71A along line B–B′.

Harvesting/growing chip 9200 comprises elastomer portion 9202 overlyingglass substrate 9204. Glass substrate 9204 computes three etched wells9206 a, 9206 b and 9206 c. Placement of elastomer portion 9202 overglass substrate 9204 thus defines three corresponding chambers in fluidcommunication with each other through flow channels 9208. The flow ofmaterials through flow channels 9208 is controlled by valves 9210defined by the overlap of control lines 9212 over control channels 9208.

During operation of growth/harvesting chip 9200, valves 9210 areinitially activated to prevent contact between the contents of chambers9206 a, 9206 b and 9206 c. Chambers 9206 a, 9206 b and 9206 c are thenseparately charged through wells 9214 with different materials foreffecting crystallization. For example, chamber 9206 a may be chargedwith a protein solution, chamber 9206 b may be charged with acrystallizing agent, and chamber 9206 c may be charged with a cryogen.

The first control line 9212 may then be deactivated to open valve 9210a, and thereby allowing diffusion of protein solution and crystallizingagent. Upon formation of a crystal 9216, the remaining control lines9212 may be deactivated to allow the diffusion of cryogen from chamber9206 c to preserve the crystal 9216.

Next, the entire chip 9200 may be mounted in an x-ray diffractionapparatus, with x-ray beam 9218 applied from source 9220 against crystal9216 with diffraction sensed by detector 9222. As shown in FIG. 71B, thegeneral location of wells 9206 corresponds to regions of reducedthickness of both elastomer portion 9202 and underlying glass portion9204. In this manner, radiation beam 9218 is required to traverse aminimum amount of elastomer and glass material prior to and subsequentto encountering crystal 9216, thereby reducing the deleterious effect ofnoise on the diffracted signal received.

While one example of a protein growth/harvesting chip has been describedabove in connection with FIGS. 71A–B, embodiments in accordance with thepresent invention are not limited to this particular structure. Forexample, while the embodiment of the current embodiment that isdescribed utilizes a glass substrate in which microchambers have beenetched, fabrication of microfluidic structures in accordance with thepresent invention is not limited to the use of glass substrates.Possible alternatives for fabricating features in a substrate includeinjection molding of plastics, hot embossing of plastics such as PMMA,or fabricating wells utilizing a photocurable polymer such as SU8photoresist. In addition, features could be formed on a substrate suchas glass utilizing laser ablation, or features could be formed byisotropic or aniosotropic etching of a substrate other than glass, suchas silicon.

Potential advantages conferred by alternative fabrication methodsinclude but are not limited to, more accurate definition of featuresallowing for more dense integration, and ease of production (e.g. hotembossing). Moreover, certain materials such as carbon based plasticsimpose less scattering of X-rays, thereby facilitating collection ofdiffraction data directly from a chip.

An embodiment of a microfluidic structure facilitating crystal growthand analysis comprises an elastomer portion bearing a recess on a lowersurface. A substrate is in contact with the lower surface of theelastomer portion to define a first microfluidic chamber, a secondmicrofluidic chamber, and a third microfluidic chamber, the first,second, and third microfluidic chambers in fluid communication through aflow channel defined between elastomer portion and the substrate. Thefirst chamber may be primed with a target material solution, the secondchamber may be primed with a crystallizing agent, and the third chambermay be primed with a cryogen, such that crystals formed in the structureby diffusion of the crystallizing agent and the target solution may bepreserved through a reduction in temperature afforded by introduction ofthe cryogen.

e. Crystal Off-Loading Methods

An additional possibility for the harvesting of crystals is to have amethod of off-loading from chip. Off-loading could be performed oncecrystals have formed, or alternatively, prior to incubation. Theseoff-loaded crystals could then be used to seed macroscopic reactions, orbe extracted and mounted in a cryo-loop. If a method for the addition ofcryogen was also developed, the crystals could be flash frozen andmounted directly into the x-ray beam.

8. Micro-Free Interface Diffusion

Since it may be difficult to determine, a priori, which thermodynamicconditions will induce crystallization, a screening method should sampleas much of phase-space (as many conditions) as possible. This can beaccomplished by conducting a plurality of assays, and also through thephase space sampled during the evolution of each assay in time. Oneconventional method that is particularly effective at sampling a widerange of conditions is macroscopic free-interface diffusion. Thistechnique requires the creation of a well-defined fluidic interfacebetween two or more solutions, typically the protein stock, and theprecipitating agent, and the subsequent equilibration of the twosolutions via a diffusive process. As the solutions diffuse into oneanother, a gradient is established along the diffusion path, and acontinuum of conditions is simultaneously sampled. Since there is avariation in the conditions, both in space, and in time, informationregarding the location and time of crystal formation may be used infurther optimization. FIGS. 71A–71D are simplified schematic diagramsplotting concentration versus distance for a solution A and a solution Bin contact along a free interface. FIGS. 71A–D show that over time, acontinuous and broad range of concentration profiles of the twosolutions is ultimately created.

Despite the efficiency of macroscopic free-interface diffusiontechniques, technical difficulties have rendered it unsuitable for highthroughput screening applications, and it is not widely used in thecrystallographic community for several reasons. First, the fluidicinterfaces are typically established by dispensing the solutions into anarrow container; such as a capillary tube or a deep well in a cultureplate. FIGS. 72A–B show simplified cross-sectional views of theattempted formation of a macroscopic free-interface in a capillary tube9300. The act of dispensing a second solution 9302 into a first solution9304 creates convective mixing and results in a poorly defined fluidicinterface 9306.

Moreover, the solutions may not be sucked into a capillary serially toeliminate this problem. FIGS. 73A–B show the mixing, between a firstsolution 9400 and a second solution 9402 in a capillary tube 9404 thatwould result due to the parabolic velocity distribution of pressuredriven Poiseuille flow, resulting in a poorly defined fluidic interface9406. Furthermore, the container for a macro free-interfacecrystallization regime must have dimensions making them accessible to apipette tip or dispensing tool, and necessitating the use of large(10–100 μl) volumes of protein and precipitant solutions.

In order to avoid unwanted convective mixing, care must be taken bothduring dispensing and during crystal incubation. For this reasoncumbersome protocols are often used to define a macro free-interface.For example, freezing one solution prior to the addition of the second.Moreover, two solutions of differing density will mix by gravity inducedconvection if they are not stored at the proper orientation,additionally complicating the storage of reactions. This is shown inFIGS. 74A–C, wherein over time first solution 9500 having a densitygreater than the density of second solution 9502 merely sinks to form astatic bottom layer 9504 that is not conducive to formation of adiffusion gradient along the length of a capillary tube.

In accordance with embodiments of the present invention, acrystallization technique analogous to traditional macro-free interfacediffusion, called gated micro free interface diffusion (Gated μ-FID),has been developed. Gated μ-FID retains the efficient sampling of phasespace achieved by macroscopic free interface diffusion techniques, withthe added advantages of parsimonious use of sample solutions, ease ofset-up, creation of well defined fluidic interfaces, control overequilibration dynamics, and the ability to conduct high-throughputparallel experimentation. These advantages are made possible by a numberof features of the instant invention.

Microfluidics enables the handling of fluids on the sub-nanoliter scale.Consequently, there is no need to use large containment chambers, andhence, assays may be performed on the nanoliter, or subnanoliter scale.The utilization of extremely small volumes allows for thousands ofassays to be performed to consume the same sample volume required forone macroscopic free-interface diffusion experiment. This reduces costlyand time-consuming amplification and purification steps, and makespossible the screening of proteins that are not easily expressed, andhence must be purified from a bulk sample.

Microfluidics further offers savings in preparation times, as hundreds,or even thousands of assays may be performed simultaneously. The use ofscaleable metering techniques as previously described, allow forparallel experimentation to be conducted without increased complexity incontrol mechanisms.

The elastomeric material from which microfluidic structures inaccordance with embodiments of the present invention may be formed, isrelatively permeable to certain gases. This gas permeability propertymay be exploited utilizing the technique of pressurized out-gas priming(POP) to form well-defined, reproducible fluidic interfaces.

FIG. 75A shows a cross-sectional view of a flow channel 9600 of amacrofluidic device in accordance with an embodiment of the presentinvention. Flow channel 9600 is separated into two halves by actuatedvalve 9602. Prior to the introduction of material, flow channel 9600contains a gas 9604.

FIG. 75B shows the introduction of a first solution 9606 to first flowchannel portion 9600 a under pressure, and the introduction of a secondsolution 9608 to second flow channel portion 9600 b under pressure.Because of the gas permeability of the surrounding elastomer material9607, gas 9604 is displaced by the incoming solutions 9608 and 9610 andout-gasses through elastomer 9607.

As shown in FIG. 75C, the pressurized out-gas priming of flow channelportions 9600 a and 9600 b allows uniform filling of these dead-endedflow channel portions without air bubbles. Upon deactuation of valve9602 as shown in FIG. 75D, a well-defined fluidic interface 9612 iscreated, allowing for formation of a diffusion gradient.

To summarize, conventional macro free-interface techniques employcapillary tubes or other containers having dimensions on the order ofmm. By contrast, the fluidic interface in accordance with embodiments ofthe present invention is created in a microchannel having dimensions onthe order of μm. At such small dimensions, unwanted convection issuppressed due to viscosity effects, and mixing is dominated bydiffusion. A well-defined fluidic interface may thus be establishedwithout significant undesirable convective mixing.

An embodiment of a method for crystallizing a target material comprisesdefining a first microfluidic chamber, priming the first microfluidicchamber with a solution including the target material, defining a secondmicrofluidic chamber, and priming the second microfluidic chamber with asolution including a crystallizing agent. The first microfluidic chamberis placed into fluid communication with the second microfluidic chamberto define a microfluidic free interface between the target material andthe crystallizing agent. Diffusion is permitted to occur between thetarget material and the crystallizing agent, such that a change in thesolvent environment of the target material causes the target material toform a crystal.

9. Temporal Control Over Equilibration

The growth and quality of crystals is determined not only bythermodynamic conditions explored during the equilibration, but also bythe rate at which equilibration takes place. It is therefore potentiallyvaluable to control the dynamics of equilibration.

In conventional crystallization methods, course control only over thedynamics of equilibration may be available through manipulation ofinitial conditions. For macroscopic free interface diffusion, oncediffusion begins, the experimenter has no control over the subsequentequilibration rate. For hanging drop experiments, the equilibration ratemay be changed by modifying the size of the initial drop, the total sizeof the reservoir, or the temperature of incubation. In microbatchexperiments, the rate at which the sample is concentrated may be variedby manipulating the size of the drop, and the identity and amount of thesurrounding oil. Since the equilibration rates depend in a complicatedmanner on these parameters, the dynamics of equilibration may only bechanged in a coarsely manner. Moreover, once the experiment has begun,no further control over the equilibration dynamics is available.

By contrast, the fluidic interface in a gated μ-FID experiment may becontrolled by opening or closing the interface valve, allowing preciseregulation of the equilibration dynamics. For example, FIG. 76A shows asimplified plan view of two microfluidic chambers 9700 and 9702, whosecommunication through flow channel 9704 is controlled by valve 9706.FIG. 76B plots the concentration of the first solution in the firstchamber over time, where the valve is actuated at a duty cycle of 100%.FIG. 76C plots the concentration of the first solution in the firstchamber over time, where the valve is actually at a duty cycle of 50%that in FIG. 76B. Inspection of FIG. 76C indicates that it takes twiceas long for the concentration of the first solution in chamber A to fallto one-half its original value (t^(50%) _(1/2)=2t^(100%) _(1/2)).

In accordance with an alternative embodiment of the present invention,rather than being opened and closed on a regular basis according to aduty cycle, the connecting valve may be closed at an intermediate timeor over an irregular series of actuations, thereby halting equilibrationonce a favorable condition has been achieved.

An embodiment of a method of exercising temporal control over diffusionbetween two fluids comprises providing a microfluidic flow channel in anelastomer material, a membrane portion of the elastomer materialpositioned within the flow channel to define a valve. A first portion ofthe flow channel on one side of the membrane is primed with a firstfluid. A second portion of the flow channel on the opposite side of theflow channel is primed with a second fluid. The elastomer membrane isrepeatedly moved into and out of the flow channel over time to allowdiffusion between the first fluid and the second fluid across the valve.

The equilibration rate may also be controlled by manipulation of thedimensions of the reaction chambers and of the connecting channels. Togood approximation, the time required for equilibration varies as thesquare of the required diffusion length. The equilibration rate alsodepends on the cross-sectional area of the connecting channels. Therequired time for equilibration may therefore be controlled by changingboth the length, and the cross-sectional area of the connectingchannels.

FIG. 77A shows three sets of pairs of compound chambers 9800, 9802, and9804, each pair connected by microchannels 9806 of a different lengthΔx. FIG. 77B plots equilibration time versus equilibration distance.FIG. 77B shows that the required time for equilibration of the chambersof FIG. 77A varies as the square of the length of the connectingchannels.

FIG. 78 shows four compound chambers 9900, 9902, 9904, and 9906, eachhaving different arrangements of connecting microchannel(s) 9908.Microchannels 9908 have the same length, but differ in cross-sectionalarea and/or number of connecting channels. The rate of equilibration maythus be increased/decreased by decreasing/increasing the cross-sectionalarea, for example by decreasing/increasing the number of connectingchannels or the dimensions of those channels.

Varying the equilibration rate by changing the geometry of connectingchannels may be used on a single device to explore the effect ofequilibration dynamics on crystal growth. FIGS. 79A–D show an embodimentin which a gradient of concentrations, initially established by thepartial diffusive equilibration of two solutions from a micro-freeinterface, can be maintained by the actuation of containment valves.

FIG. 79A shows flow channel 10000 that is overlapped at intervals by aforked control channel 10002 to define a plurality of chambers (A–G)positioned on either side of a separately-actuated interface valve10004. FIG. 79B plots solvent concentration at an initial time, wheninterface valve 10004 is actuated and a first half 10000 a of the flowchannel has been mixed with a first solution, and a second half 10000 bof the flow channel has been primed with a second solution. FIG. 79Cplots solvent concentration at a subsequent time T₁, when controlchannel 10002 is actuated to define the seven chambers (A–G), whichcapture the concentration gradient at that particular point in time.FIG. 79D plots relative concentration of the chambers (A–G) at time T₁.

In the embodiment shown in FIG. 79A, actuation of the forked controlchannel simultaneously creates the plurality of chambers A–G. However,this is not required, and in alternative embodiments of the presentinvention multiple control channels could be utilized to allowindependent creation of chambers A–G at different time intervals,thereby allow additional diffusion to occur after an initial set ofchambers are created immediately adjacent to the free interface.

An embodiment of a method of capturing a concentration gradient betweentwo fluids comprises providing a first fluid on a first side of anelastomer membrane present within a microfluidic flow channel, andproviding a second fluid on a second side of the elastomer membrane. Theelastomer membrane is displaced from the microfluidic flow channel todefine a microfluidic free interface between the first fluid and thesecond fluid. The first fluid and the second fluid are allowed todiffuse across the microfluidic free interface. A group of elastomervalves positioned along the flow channel at increasing distances fromthe microfluidic free interface are actuated to define a succession ofchambers whose relative concentration of the first fluid and the secondfluid reflects a time of diffusion across the microfluidic freeinterface.

10. Chip Holder

As previously illustrated, embodiments of microfluidic devices inaccordance with the present invention may utilize on-chip reservoirs orwells. However, in a microfluidic device requiring the loading of alarge number of solutions, the use of a corresponding large number ofinput tubes with separate pins for interfacing each well may beimpractical given the relatively small dimensions of the fluidic device.In addition, the automated use of pipettes for dispensing small volumesof liquid is known, and thus it therefore may prove easiest to utilizesuch techniques to pipette solutions directly on to wells present on theface of a chip.

Capillary action may not be sufficient to draw solutions from on-chipwells into active regions of the chip, particularly where dead-endedchambers are to be primed with material. In such embodiments, one way ofloading materials into the chip is through the use of externalpressurization. Again however, the small dimensions of the devicecoupled with a large number of possible material sources may renderimpractical the application of pressure to individual wells through pinsor tubing.

Accordingly, FIG. 80 shows an exploded view of a chip holder 11000 inaccordance with one embodiment of the present invention. Bottom portion11002 of chip holder 11000 includes raised peripheral portion 11004surrounding recessed area 11006 corresponding in size to the dimensionsof chip 11008, allowing microfluidic chip 11008 to be positionedtherein. Peripheral region 11004 further defines screw holes 11010.

Microfluidic device 11008 is positioned within recessed area 11006 ofbottom portion 11002 of chip holder 11000. Microfluidic device 11008comprises an active region 11011 that is in fluidic communication withperipheral wells 11012 configured in first and second rows 11012 a and11012 b, respectively. Wells 11012 hold sufficient volumes of materialto allow device 11008 to function. Wells 11012 may contain, for example,solutions of crystallizing agents, solutions of target materials, orother chemical reagents such as stains. Bottom portion 11002 contains awindow 11003 that enables active region 11011 of chip 11008 to beobserved.

Top portion 11014 of chip holder 11000 fits over bottom chip holderportion 11002 and microfluidic chip 11008 positioned therein. For easeof illustration, in FIG. 80 top chip holder portion 11014 is showninverted relative to its actual position in the assembly. Top chipholder portion 11014 includes screw holes 11010 aligned with screw holes11010 of lower holder portion 11002, such that screws 11016 may beinserted through holes 11010 secure chip between portions 11002 and11014 of holder 11000. Chip holder upper portion 11014 contains a window11005 that enables active region 11011 of chip 11008 to be observed.

Lower surface 11014 a of top holder portion 11014 includes raisedannular rings 11020 and 11022 surrounding recesses 11024 and 11026,respectively. When top portion 11014 of chip holder 11000 is pressedinto contact with chip 11008 utilizing screws 11016, rings 11020 and11022 press into the soft elastomeric material on the upper surface ofchip 11008, such that recess 11024 defines a first chamber over top row11012 a of wells 11012, and recess 11026 defines a second chamber overbottom row 11012 b of wells 11012. Holes 11030 and 11032 in the side oftop holder portion 11014 are in communication with recesses 11024 and11026 respectively, to enable a positive pressure to be applied to thechambers through pins 11034 inserted into holes 11030 and 11032,respectively. A positive pressure can thus simultaneously be applied toall wells within a row, obviating the need to utilize separateconnecting devices to each well.

In operation, solutions are pipetted into the wells 11012, and then chip11008 is placed into bottom portion 11002 of holder 11000. The topholder portion 11014 is placed over chip 11008, and is pressed down byscrews. Raised annular rings 11020 and 11022 on the lower surface of topholder portion 11014 make a seal with the upper surface of the chipwhere the wells are located. Solutions within the wells are exposed topositive pressures within the chamber, and are thereby pushed into theactive area of microfluidic device.

The downward pressure exerted by the chip holder may also pose theadvantage of preventing delamination of the chip from the substrateduring loading. This prevention of delamination may enable the use ofhigher priming pressures.

The chip holder shown in FIG. 80 represents only one possible embodimentof a structure in accordance with the present invention. For example, achip holder may also include a third portion which fits over controlline outlet ports on the front or back side of the chip, therebyenabling the application of pressure to control lines to control valveactuation within the chip. In addition, while the described holderembodiment includes a window for viewing of the chip, this may not benecessary if the chip is to be removed from the holder once the chipfilling process is complete.

In still other alternative embodiments, a chip holder in accordance withthe present invention could be equipped with heating elements to providespatial and temporal temperature profile to the chip positioned therein.Such alternative embodiments would eliminate the complexity and expenseassociated with incorporating heating elements directly onto a substratethat may be disposable.

In the particular embodiment of the chip holder illustrated in FIG. 80,the top piece is pressed to the chip by turning screws. However, inalternative embodiments in accordance with the present invention, thedownward force could be applied through a press or robotic arm, therebypotentially eliminating the need for a bottom holder piece.

Furthermore, in the particular embodiment of the chip holder illustratedin FIG. 80, the seal over the wells allowing application of a positivepressure is created by pressing the raised ring into the compliant topsurface of the elastomer chip. However, in accordance with alternativeembodiments of the present invention, a seal could be created by theaddition of flexible o-rings to the chip holder. Such o-rings wouldpermit use of a chip holder with embodiments of microfluidic devicesthat feature a rigid top surface.

Finally, it is important to recognize that use of a chip holderstructure in accordance with embodiments of the present invention is notlimited to protein crystallization, but enables loading of a largenumber of solutions onto a microfluidic chip for performance of avariety of applications.

An embodiment of a structure for applying pressure to a elastomericmicrofluidic device in accordance with the present invention comprises,a holder portion including a continuous raised rim on a lower surfacethereof configured to contact a top surface of the microfluidic deviceand surround a plurality of material wells located therein. Contactbetween the raised rim and the top surface of the microfluidic devicedefines an airtight chamber over the material wells, an orifice incommunication with the chamber enabling application of positive pressureto the airtight chamber to drive the contents of the material wells intoan active area of the microfluidic device.

An embodiment of a method of priming a microfluidic device with a liquidmaterial in accordance with the present invention comprises loading aplurality of wells on an upper surface of a microfluidic device with aliquid material. A holder piece is biased against the upper surface suchthat a continuous raised rim of the holder piece presses against theupper surface surrounding the wells, such that a chamber is created overthe wells. A positive pressure is applied to the airtight chamber todrive the material from the wells into an active area of the elastomericmicrofluidic structure.

An embodiment of a method of actuating a valve within a microfluidicelastomer device comprises applying a holder piece having a continuousraised rim against a surface of a microfluidic device having a pluralityof control line outlets to create a chamber over the outlets. A positiveor negative pressure is applied to the airtight chamber to control apressure within the control line and thereby actuate a elastomeric valvemembrane of the microfluidic device that is in communication with thecontrol line.

11. Target Materials

Typical targets for crystallization are diverse. A target forcrystallization may include but is not limited to: 1) biologicalmacromolecules (cytosolic proteins, extracellular proteins, membraneproteins, DNA, RNA, and complex combinations thereof), 2) pre- andpost-translationally modified biological molecules (including but notlimited to, phosphorylated, sulfolated, glycosylated, ubiquitinated,etc. proteins, as well as halogentated, abasic, alkylated, etc. nucleicacids); 3) deliberately derivatized macromolecules, such as heavy-atomlabeled DNAs, RNAs, and proteins (and complexes thereof),selenomethionine-labeled proteins and nucleic acids (and complexesthereof), halogenated DNAs, RNAs, and proteins (and complexes thereof),4) whole viruses or large cellular particles (such as the ribosome,replisome, spliceosome, tubulin filaments, actin filaments, chromosomes,etc.), 5) small-molecule compounds such as drugs, lead compounds,ligands, salts, and organic or metallo-organic compounds, and 6)small-molecule/biological macromolecule complexes (e.g., drug/proteincomplexes, enzyme/substrate complexes, enzyme/product complexes,enzyme/regulator complexes, enzyme/inhibitor complexes, and combinationsthereof). Such targets are the focus of study for a wide range ofscientific disciplines encompassing biology, biochemistry, materialsciences, pharmaceutics, chemistry, and physics.

A nonexclusive listing of possible protein modifications is as follows:5′ dephospho; Desmosine (from Lysine); decomposed carboxymethylatedMethionine; Omithine (from Arginine); Lysinoalanine (from Cysteine);Lanthionine (from Cysteine); Dehydroalanine (from Cysteine); Homoserineformed from Met by CNBr treatment; Dehydration (—H2O); S-gamma-Glutamyl(crosslinked to Cysteine); O-gamma-Glutamyl-(Crosslink to Serine);Serine to Dehydroalanine; Alaninohistidine (Serine crosslinked to thetaor pi carbon of Histidine); Pyroglutamic Acid formed fromGln;N-pyrrolidone carboxyl (N terminus); Nalpha-(gamma-Glutamyl)-lysine; N-(beta-Aspartyl)-Lysine (Crosslink);3,3′,5,5′-TerTyr (Crosslink); Disulphide bond formation (Cystine);S-(2-Histidyl)-(Crosslinked to Cysteine); S-(3-Tyr) (Crosslinked toCysteine); 3,3′-BiTyr (Crosslink); IsodiTyr (Crosslink); Allysine (fromLysine); Amide formation (C terminus); Deamidation of Asparagine andGlutamine to Aspartate and Glutamate; Citruline (from Arginine);Syndesine (from Lysine); Methylation (N terminus, N epsilon of Lysine, Oof Serine, Threonine or C terminus, N of Asparagine);delta-Hydroxy-allysine (from Lysine); Hydroxylation (of delta C ofLysine, beta C of Tryptophan, C3 or C4 of Proline, beta C of Aspartate);Oxidation of Methionine (to Sulphoxide); Sulfenic Acid (from Cysteine);Pyruvoyl-(Serine); 3,4-Dihydroxy-Phenylalanine (from Tyrosine) (DOPA);Sodium; Ethyl; N,N dimethylation (of Arginine or Lysine);2,4-BisTrp-6,7-dione (from Tryptophan); Formylation (CHO); 6,7 Dione(from Tryptophan); 3,4,6-Trihydroxy-Phenylalanine (from Tyrosine)(TOPA); 3,4-Dihydroxylation (of Proline); Oxidation of Methionine (toSulphone); 3-Chlorination (of Tyrosine with 35Cl); 3-Chlorination (ofTyrosine with 37Cl); Potassium; Carbamylation; Acetylation (N terminus,N epsilon of Lysine, O of Serine) (Ac); N-Trimethylation (of Lysine);gamma Carboxylation of Glutamate or beta Carboxylation of Aspartate;disodium; Nitro (NO2); t-butyl ester(OtBu) and t-butyl (tBu); Glycyl(-G-,-Gly-); Carboxymethyl (on Cystine); sodium+potassium;Selenocysteine (from Serine); 3,5-Dichlorination (of Tyrosine with35Cl); Dehydroalanine (Dha); 3,5-Dichlorination (of Tyrosine withmixture of 35Cl and 37Cl)); Pyruvate; Acrylamidyl or Acrylamide adduct;Sarcosyl; Alanyl (-A-, -Ala-); Acetamidomethyl (Acm); 3,5-Dichlorination(of Tyrosine with 37Cl);S-(sn-1-Glyceryl) (on Cysteine); Glycerol Ester(on Glutamic acid side chain); Glycine (G, Gly); Beta mercaptoethanoladduct; Phenyl ester (OPh) (on acidic); 3-Bromination (of Tyrosine with79Br); Phosphorylation (O of Serine, Threonine, Tyrosine and Aspartate,N epsilon of Lysine); 3-Bromination (of Tyrosine with 81Br);Sulphonation (SO3H) (of PMC group); Sulphation (of O of Tyrosine);Cyclohexyl ester (OcHex); Homoseryl lactone; Dehydroamino butyric acid(Dhb); Gamma Aminobutyryl; 2-Aminobutyric acid (Abu); 2-Aminoisobutyricacid (Aib); Diaminopropionyl; t-butyloxymethyl (Bum);N-(4-NH2-2-OH-butyl)-(of Lysine) (Hypusine); Seryl (-S-, -Ser-);t-butylsulfenyl (StBu); Alanine (A, Ala); Sarcosine (Sar); Amisyl;Benzyl (Bzl) and benzly ester (OBzl); 1,2-ethanedithiol (EDT);Dehydroprolyl; Triflouroacetyl (TFA); N-hydroxysuccinimide (ONSu, OSu);Prolyl (-P-, -Pro-); Valyl (-V-, -Val-); Isovalyl (-I-,-Iva-);t-Butyloxycarbonyl (tBoc); Threoyl (-T-, -Thr-);Homoseryl (-Hse-);Cystyl (-C-, -Cys-); Benzoyl (Bz); 4-Methylbenzyl (Meb); Serine (S,Ser);HMP (hydroxymethylphenyl) linker; Thioanisyl; Thiocresyl;Diphthamide (from Histidine); Pyroglutamyl; 2-Piperidinecarboxylic acid(Pip); Hydroxyprolyl (-Hyp-); Norleucyl (-Nle-); Isoleucyl (-I-,-Ile-);Leucyl (-L-, -Leu-); Omithyl (-Om-); Asparagyl (-N-, -Asn-);t-amyloxycarbonyl (Aoc); Proline (P, Pro); Aspartyl (-D-, -Asp-);Succinyl; Valine (V, Val); Hydroxybenzotriazole ester (HOBt);Dimethylbenzyl (diMeBzl); Threonine (T, Thr); Cysteinylation;Benzyloxymethyl (Bom); p-methoxybenzyl (Mob, Mbzl); 4-Nitrophenyl,p-Nitrophenyl (ONp); Cysteine (C, Cys); Chlorobenzyl (ClBzl); lodination(of Histidine[C4] or Tyrosine[C3]); Glutamyl (-Q-, -Gln-); N-methylLysyl; Lysyl (-K-, -Lys-); O-Methyl Aspartamyl; Glutamyl (-E-, -Glu-); Nalpha -(gamma-Glutamyl)-Glu; Norleucine (Nle); Hydroxy Aspartamyl;Hydroxyproline (Hyp); bb-dimethyl Cystenyl; Isoleucine (I, Ile); Leucine(L, Leu); Methionyl (-M-, -Met-); Asparagine (N, Asn); Pentoses (Ara,Rib, Xyl); Aspartic Acid (D, Asp); Dmob (Dimethoxybenzyl);Benzyloxycarbonyl (Z); Adamantyl (Ada); p-Nitrobenzyl ester (ONb);Histidyl (-H-, -His-); N-methyl Glutamyl; O-methyl Glutamyl; HydroxyLysyl (-Hyl-); Methyl Methionyl; Glutamine (Q, Gln); AminoethylCystenyl; Pentosyl; Deoxyhexoses (Fuc, Rha); Lysine (K, Lys); Aminoethylcystenyl (-AECys-); 4-Glycosyloxy-(pentosyl, C5) (of Proline); MethionylSulfoxide; Glutamic Acid (E, Glu); Phenylalanyl-(-F-, -Phe-); PyridylAlanyl; Flourophenylalanyl; 2-Nitrobenzoyl (NBz); Methionine (M, Met);3-methyl Histidyl; 2-Nitrophenylsulphenyl (Nps); 4-Toluenesulphonyl(Tosyl, Tos); 3-nitro-2-pyridinesulfenyl (Npys); Histidine (H, His);3,5-Dibromination (of Tyrosine with 79Br); Arginyl (-R-, -Arg-);Citrulline; 3,5-Dibromination (of Tyrosine with mixture of 79Br and81Br); Dichlorobenzyl (Dcb); 3,5-Dibromination (of Tyrosine with 81Br);Carboxyamidomethyl Cystenyl; Carboxymethyl Cystenyl; Methylphenylalanyl;Hexosamines (GalN, GlcN); Carboxymethyl cysteine (Cmc); N-Glucosyl (Nterminus or N epsilon of Lysine) (Aminoketose); O-Glycosyl-(to Serine orThreonine); Hexoses (Fru, Gal, Glc, Man); Inositol; MethionylSulphone;Tyrosinyl (-Y-, -Tyr-); Phenylalanine (F, Phe); 2,4-dinitrophenyl (Dnp);Pentaflourophenyl (Pfp); Diphenylmethyl (Dpm); Phospho Seryl;2-Chlorobenzyloxycarbonyl (ClZ); Napthyl acetyl; Isopropyl Lysyl;N-methyl Arginyl; Ethaneditohiol/TFA cyclic adduct; Carboxy Glutamyl(Gla); Acetamidomethyl Cystenyl; Acrylamidyl Cystenyl; Arginine (R,Arg); N-Glucuronyl (N terminus); delta-Glycosyloxy-(of Lysine) orbeta-Glycosyloxy-(of Phenylalanine or Tyrosine); 4-Glycosyloxy-(hexosyl,C6) (of Proline); Benzyl Seryl; N-methyl Tyrosinyl;p-Nitrobenzyloxycarbonyl (4Nz); 2,4,5-Trichlorophenyl;2,4,6-trimethyloxybenzyl (Tmob); Xanthyl (Xan); Phospho Threonyl;Tyrosine (Y, Tyr); Chlorophenylalanyl; Mesitylene-2-sulfonyl (Mts);Carboxymethyl Lysyl; Tryptophanyl (-W-, -Trp-); N-Lipoyl-(on Lysine);Matrix alpha cyano MH+; Benzyl Threonyl; Benzyl Cystenyl; NapthylAlanyl; Succinyl Aspartamyl; Succinimidophenyl carb.; HMP(hydroxymethylphenyl)/TFA adduct; N-acetylhexosamines (GalNAc, GlcNAc);Tryptophan (W, Trp); Cystine ((Cys)2); Farnesylation; S-Farnesyl-;Myristoleylation (myristoyl with one double bond); PyridylethylCystenyl; Myristoylation; 4-Methoxy-2,3,6-trimethylbenzenesulfonyl(Mtr); 2-Bromobenzyloxycarbonyl (BrZ); Formyl Tryptophanyl; BenzylGlutamyl; Anisole Adducted Glutamyl; S-cystenyl Cystenyl;9-Flourenylmethyloxycarbonyl (Fmoc); Lipoic acid (amide bond to lysine);Biotinylation (amide bond to lysine); Dimethoxybenzhydryl (Mbh);N-Pyridoxyl (on Lysine); Pyridoxal phosphate (Schiff Base formed tolysine); Nicotinyl Lysyl; Dansyl (Dns);2-(p-biphenyl)isopropyl-oxycarbonyl (Bpoc); Palmitoylation;“Triphenylmethyl (Trityl, Trt)”; Tyrosinyl Sulphate; Phospho Tyrosinyl;Pbf (pentamethyldihydrobenzofuransulfonyl); 3,5-Diiodination (ofTyrosine); 3,5-di-I″;N alpha -(gamma-Glutamyl)-Glu2;O-GlcNAc-1-phosphorylation (of Serine);“2,2,5,7,8-Pentamethylchroman-6-sulphonyl (Pmc)”; Stearoylation;Geranylgeranylation; S-Geranylgeranyl; 5′phos dCytidinyl; iodoTyrosinyl; Aldohexosyl Lysyl; Sialyl; N-acetylneuraminic acid (Sialicacid, NeuAc, NANA, SA); 5′phos dThymidinyl; 5′phos Cytidinyl;Glutathionation; O-Uridinylylation (of Tyrosine); 5′phos Uridinyl;S-famesyl Cystenyl; N-glycolneuraminic acid (NeuGc); 5′phos dAdenosyl;O-pantetheinephosphorylation (of Serine); SucPhencarb Lysyl; 5′phosdGuanosyl; 5′phos Adenosinyl; O-5′-Adenosylation (of Tyrosine);4′-Phosphopantetheine; GL2; S-palmityl Cystenyl; 5′phos Guanosyl;Biotinyl Lysyl; Hex-HexNAc; N alpha-(gamma-Glutamyl)-Glu3; DioctylPhthalate; PMC Lysyl; Aedans Cystenyl; Dioctyl Phthalate Sodium Adduct;di-iodo Tyrosinyl; PMC Arginyl; S-Coenzyme A; AMP Lysyl;3,5,3′-Triiodothyronine (from Tyrosine);S-(sn-1-Dipalmitoyl-glyceryl)-(on Cysteine); S-(ADP-ribosyl)-(onCysteine); N-(ADP-ribosyl)-(on Arginine); O-ADP-ribosylation (onGlutamate or C terminus); ADP-rybosylation (from NAD); S-Phycocyanobilin(on Cysteine); S-Heme (on Cysteine); N theta-(ADP-ribosyl) diphthamide(of Histidine); NeuAc-Hex-HexNAc; MGDG; O-8 alpha-Flavin [FAD])-(ofTyrosine); S-(6-Flavin [FAD])-(on Cysteine); N theta and Npi-(8alpha-Flavin) (on Histidine); (Hex)3-HexNAc-HexNAc;(Hex)3-HexNAc-(dHex)HexNAc.

A nonexclusive listing of possible nucleic acid modifications, such asbase-specific, sugar-specific, or phospho-specific is as follows:halogenation (F, Cl, Br, I); Abasic sites; Alkylation; Crosslinkableadducts such as thiols or azides; Thiolation; Deamidation;Fluorescent-group labeling, and glycosylation.

A nonexclusive listing of possible heavy atom derivatizing agents is asfollows: potassium hexachloroiridate (III); Potassium hexachloroiridate(IV); Sodium hexachloroiridate (IV); Sodium hexachloroiridate (III);Potassium hexanitritoiridate (III); Ammnoium hexachloroiridate (III);Iridium (III) chloride; Potassium hexanitratoiridate (III); Iridium(III) bromide; Barium (II) chloride; Barium (II) acetate; Cadmium (II)nitrate; Cadmium (II) iodide; Lead (II) nitrate; Lead (II) acetate;Trimethyl lead (IV) chloride; Trimethyl lead (IV) acetate; Ammoniumhexachloro plumbate (IV); Lead (II) chloride; Sodium hexachlororhodiate(III); Strontium (II) acetate; Disodium thiomalonato aurate (I);Potassium dicyano aurate (I); Sodium dicyano aurate (I); Sodiumthiosulphato aurate (III); Potassium tetracyano aurate (III); Potassiumtetrachloro aurate (III); Hydrogen tetrachloro aurate (III); Sodiumtetrachloro aurate (III); Potassium tetraiodo aurate (III); Potassiumtetrabromo aurate (III); (acetato-o)methylmercury; Methyl (nitrato-o)mercury; Chloromethylmercury; lodomethylmercury; Chloroethylmercury;Methyl mercury cation; Triethyl (m3-phosphato(3-)-0,0′,0″)tri mercuryeth; [3-[(aminocarbonyl)amino]-2-methoxypropyl]chlorome; 1,4diacetoxymercury 2-3 dimethoxy butane; Meroxyl mercuhydrin; Tetrakis(acetoxy mercuri)-methane; 1,4-bis(chloromercuri)-2,3-butanediol; Ethyldiacetoxymercurichloro acetate (dame); Mercuric (II) oxide; Methylmercuri-2-mercaptoethanol; 3,6 bis (mercurimethyl dioxane acetate);Ethyl mercury cation; Billman's dimercurial; Para chloromercury phenylacetate (pcma); Mercury phenyl glyoxal (mpg); Thiomersal, ethyl mercurythiosalicylate [emts]; 4-chloromercuribenesulphonic acid; 2,6dichloromercuri-4-nitrophenol (dcmnp);[3-[[2(carboxymethoxy)benzoyl]mino-2 methoxy prop; Parachloromercurybenzoate (pcmb), 4-chloromercury; (acetato-o)phenyl mercury; Phenylmercuri benzoate (pmb); Para hydroxy mercuri benzoate (phmb); Mercuricimidosuccinate/mercury succinimide; 3-hydroxymercurybenzaldehyde;2-acetoxy mercuri sulhpanilamide;3-acetoxymercuri-4-aminobenzenesulphonamide; Methyl mercuri thioclycolicacid (mmtga); 2-hydroxymercuri-tolulen-4-sulphonic acid (hmts);Acetamino phenyl mercury acetate (apma);[3-[(aminocarbonyl)amino]-3-methoxypropyl 2-chloro; Para-hydroxymercuribenzene sulphonate (phmbs); Ortho-chloromercuri phenol (ocmp);Diacetoxymercury dipopylene dioxide (dmdx); Para-acetoxymercuri aniline(pama); (4-aminophenyl) chloromercury; Aniline mercury cation;3-hydroxy-mercuri-s sulphosalicylic acid (msss); 3 or 5 hydroxymercurisalicylic acid (hmsa); Diphenyl mercury; 2,6 diacetoxymercurimethyl 1-4thioxane (dmmt); 2,5-b1s (chloromercury) furan; Ortho-chloromercurinitrophenol (ocmnp); 5-mercurydeoxyuridine monosulphate; Mercurysalicylate; [3-[[2-(carboxymethoxy)benzoyl]amino-2-methoxypro; 3,3 bis(hydroximercuri)-3-nitratomercuri pyruvic; 3-chloro mercuri pyridine;3,5 bis acetoxymercuri methyl morpholine; Ortho-mercury phenol cation;Para-carboxymethyl mercaptomercuri benzensulphonyl; Para-mercuribenzoylglucosamine; 3-acetetoxymercuri-5-nitrosalicyladehyde (msa); Ammoniumtetrachloro mercurate (II); Potassium tetrathiocyanato mercurate (II);Sodium tetrathiocyanato mercurate (II); Potassium tetraisothiocyantomercurate (II); Potassium tetraido mercurate (II); Ammoniumtetrathiocyananato mercurate (II); Potassium tetrabromo mercurate (II);Potassium tetracyano mercurate (II); Mercury (II) bromide; Mercury (II)thiocyanate; Mercury (II) cyanide; Mercury (II) iodide; Mercuric (II)chloride; Mercury (II) acetate; Mercury (I) acetate; Dichlorodiaminomercurate (II); Beta mercury-mercapto-ethylamine hydrochloride; Mercury(II) sulphate; Mercury (II) chloroanilate; Dimercuriacetate;Chloro(2-oxoethyl) mercury; Phenol mercury nitrate; Mercurymercaptoethanol; Mercury mercaptoethylamine chloride; Mercurythioglycollic acid (sodium salt);0-hydroxymercuri-p-nitrophenol/2-hydroxymercuri-4-; Para chloromercuriphenol (pcmp); Acetylmercurithiosalicylate (amts); Iodine; Potassiumiodide (iodine); 4-iodopyrazole; O-iodobenzoylglucasamine;P-iodobenzoylglucasamine; Potassium iodide/chloramine t; Ammoniumiodide; 3-isothio-cyanato-4-iodobenzene sulphonate; Potassium iodide;3′-iodo phenyltrazine; 4′-iodo phenyltrazine; Sodium iodide/iodine;Silver nitrate; Silver ( ) trinitridosulphoxylate; Tobenamed; Samarium(III) chloride; Thulium (III) chloride; Lutetium (III) chloride;Europium (III) chloride; Terbium (III) chloride; Gadolinium (III)chloride; Erbium (III) chloride; Lanthanum (III) chloride; Samarium(III) nitrate; Samarium (III) acetate; Samarium (III) cation;Praseodymium (III) chloride; Neodymium (III) chloride; Ytterbium (III)chloride; Thulium (III) sulphate; Ytterbium (III) sulphate; Gadolinium(III) sulphate; Gadolinium (III) acetate; Dysprosium (III) chloride;Erbium (III) nitrate; Holmium (III) chloride; Penta amino ruthenium(III) chloride; Cesium nitridotiroxo osmium (viii); Potassium tetraoxoosmiate; Hexa amino osmium (III) iodide; Ammonium hexachloroosmiate(IV); Osmium (III) chloride; Potassium hexachloro osmiate (IV); Cesiumtrichloro triscarbonyl osmiate (?); Dinitritodiamine platinum (II); Cisdichlorodimethylammido platinum (II); Dichlorodiammine platinum (II);Dibromodiammine platinum (II); Dichloroethylene diamine platinum (II);Potassium dicholodinitrito platinate (II); Diethylenediamene platinum(II); Potassium dioxylato platinate (II); Dichlorobis (pyridine)platinum (II); Potassium (thimethyl dibenzyloamine) platinum (?);Potassium tetrabromoplatinate (II); Potassium tetrachloro platinate(II); Potassium tetranitrito platinum (II); Potassium tetracyanoplatinate (II); Sodium tetracyano platinate (II); Potassiumtetrathiocyanato platinate (II); Ammonium tetranitrito platinate (II);Potassium tetraisocyanato platinate (II); Ammonium tetracyano platinum(II); Ammonium tetrachloro platinate (II); Potassium dinitritodioxalatoplatinate (IV); Dichlorotetraammino platinium (IV); Dibromodinitritodiammine platinium (IV); Potassium hexanitrito platinate (IV); Potassiumhexachloro platinate (IV); Potassium hexabromo platinate (IV); Sodiumhexachloroplatinate (IV); Potassium hexaiodo platinate (IV); Potassiumhexathiocyanato platinate (IV); Tetrachloro bis(pyridine) platinum (IV);Ammonium hexachloro platinate (IV); Di-mu-iodo bis(ethylenediamine) diplatinum (II) n; Potassium hexaisothiocyanato platinate (IV); Potassiumtetraiodo platinate (II); 2,2′,2″ terpyridyl platinium (II); 2hydroxyethanethiolate (2,2′,2″ terpyeidine) pla; Potassium tetranitroplatinate (II); Trimethyl platinum (II) nitrate; Sodium tetraoxo rhenate(VII); Potassium tetraoxo rhenate (VI); Potassium tetraoxo rhenate(VII); Potassium hexachloro rhenium (IV); Rhenium (III) chloride;Ammonium hexachloro rhenate (IV); Dimethyltin (II) dichloride; Thorium(IV) nitrate; Uranium (VI) oxychloride; Uranium (VI) oxynitrate; Uranium(VI) oxyacetate; Uranium (VI) oxypyrophosphate; Potassium pentafluorooxyuranate (VI); Sodium pentafluoro oxyuranate (VI); Potassiumnanofluoro dioxyuranate (VI); Sodium triacetate oxyuranate (VI); Uranium(VI) oxyoxalate; Selenocyanate anion; Sodium tungstate; Sodium12-tungstophosphate; Thallium (I) acetate; Thallium (I) fluoride;Thallium (I) nitrate; Potassium tetrachloro palladate (II); Potassiumtetrabromo palladate (II); Potassium tetracyano palladate (II);Potassium tetraiodo palladate (II); Cobalt (II) chloride.

The PDMS material from which the chip can be formed is well suited formany of these targets, particularly biological samples. PDMS is anon-reactive and biologically inert compound that allows such moleculesto maintain their appropriate shape, fold, and activity in a solublizedstate. The matrix and system can accommodate a range of target sizes andmolecular weights, from a few hundred Daltons to the mega-Dalton regime.Biological targets, from small proteins and peptides to viruses andmacromolecular complexes, fall within this range, and are generallyanywhere from 3–10 kDa to >1–2 MDa in size.

12. Solute/Reagent Types

During crystallization screening, a large number of chemical compoundsmay be employed. These compounds include salts, small and largemolecular weight organic compounds, buffers, ligands, small-moleculeagents, detergents, peptides, crosslinking agents, and derivatizingagents. Together, these chemicals can be used to vary the ionicstrength, pH, solute concentration, and target concentration in thedrop, and can even be used to modify the target. The desiredconcentration of these chemicals to achieve crystallization is variable,and can range from nanomolar to molar concentrations. A typicalcrystallization mix contains set of fixed, but empirically-determined,types and concentrations of ‘precipitants’, buffers, salts, and otherchemical additives (e.g., metal ions, salts, small molecular chemicaladditives, cryo protectants, etc.). Water is a key solvent in manycrystallization trials of biological targets, as many of these moleculesmay require hydration to stay active and folded.

As described above in connection with the pressurized out-gas priming(POP) technique, the permeability of PDMS to gases, and thecompatibility of solvents with PDMS may be a significant factor indeciding upon precipitating agents to be used.

‘Precipitating’ agents act to push targets from a soluble to insolublestate, and may work by volume exclusion, changing the dielectricconstant of the solvent, charge shielding, and molecular crowding.Precipitating agents compatible with the PDMS material of certainembodiments of the chip include, but are not limited to, non-volatilesalts, high molecular weight polymers, polar solvents, aqueoussolutions, high molecular weight alcohols, divalent metals.

Precipitating compounds, which include large and small molecular weightorganics, as well as certain salts are used from under 1% to upwards of40% concentration, or from <0.5M to greater than 4M concentration. Wateritself can act in a precipitating manner for samples that require acertain level of ionic strength to stay soluble. Many precipitants mayalso be mixed with one another to increase the chemical diversity of thecrystallization screen. The microfluidics devices described in thisdocument are readily compatible with a broad range of such compounds.Moreover, many precipitating agents (such as long- and short-chainorganics) are quite viscous at high concentrations, presenting a problemfor most fluid handling devices, such as pipettes or robotic systems.The pump and valve action of microfluidics devices in accordance withembodiments of the present invention enable handling of viscous agents.

An investigation of solvent/precipitating agent compatibility withparticular elastomer materails may be conducted to identify optimumcrystallizing agents, which may be employed develop crystallizationscreening reactions tailored for the chip that are more effective thanstandard screens.

A nonexclusive list of salts which may be used as precipitants is asfollows: Tartrate (Li, Na, K, Na/K, NH4); Phosphate (Li, Na, K, Na/K,NH4); Acetate (Li, Na, K, Na/K, Mg, Ca, Zn, NH4); Formate (Li, Na, K,Na/K, Mg, NH4); Citrate (Li, Na, K, Na/K, NH4); Chloride (Li, Na, K,Na/K, Mg, Ca, Zn, Mn, Cs, Rb, NH4); Sulfate (Li, Na, K, Na/K, NH4);Malate (Li, Na, K, Na/K, NH4); Glutamate (Li, Na, K, Na/K, NH4.

A nonexclusive list of organic materials which may be used asprecipitants is as follows: PEG 400; PEG 1000; PEG 1500; PEG 2k; PEG3350; PEG 4k; PEG 6k; PEG 8k; PEG 10k; PEG 20k; PEG-MME 550; PEG-MME750; PEG-MME 2k; PEG-MME 5k; PEG-DME 2k; Dioxane; Methanol; Ethanol;2-Butanol; n-Butanol; t-Butanol; Jeffamine M-600; Isopropanol;2-methyl-2,4-pentanediol; 1,6 hexanediol.

Solution pH can be varied by the inclusion of buffering agents; typicalpH ranges for biological materials lie anywhere between values of3.5–10.5 and the concentration of buffer, generally lies between 0.01and 0.25 M. The microfluidics devices described in this document arereadily compatible with a broad range of pH values, particularly thosesuited to biological targets.

A nonexclusive list of possible buffers is as follows: Na-Acetate;HEPES; Na-Cacodylate; Na-Citrate; Na-Succinate; Na-K-Phosphate; TRIS;TRIS-Maleate; Imidazole-Maleate; BisTrisPropane; CAPSO, CHAPS, MES, andimidizole.

Additives are small molecules that affect the solubility and/or activitybehavior of the target. Such compounds can speed crystallizationscreening or produce alternate crystal forms of the target. Additivescan take nearly any conceivable form of chemical, but are typically monoand polyvalent salts (inorganic or organic), enzyme ligands (substrates,products, allosteric effectors), chemical crosslinking agents,detergents and/or lipids, heavy metals, organo-metallic compounds, traceamounts of precipitating agents, and small molecular weight organics.

The following is a nonexclusive list of possible additives: 2-Butanol;DMSO; Hexanediol; Ethanol; Methanol; Isopropanol; sodium flouride;potassium flouride; ammonium flouride; lithium chloride anhydrous;magnesium chloride hexahydrate; sodium chloride; Calcium chloridedihydrate; potassium chloride; ammonium chloride; sodium iodide;potassium iodide; ammonium iodide; sodium thiocyanate; potassiumthiocyanate; lithium nitrate; magnesium nitrate hexahydrate; sodiumnitrate; potassium nitrate; ammonium nitrate; magnesium formate; sodiumformate; potassium formate; ammonium formate; lithium acetate dihydrate;magnesium acetate tetrahydrate; zinc acetate dihydrate; sodium acetatetrihydrate; calcium acetate hydrate; potassium acetate; ammoniumacetate; lithium sulfate monohydrate; magnesium sulfate heptahydrate;sodium sulfate decahydrate; potassium sulfate; ammonium sulfate;di-sodium tartate dihydrate; potassium sodium tartrate tetrahydrate;di-ammonium tartrate; sodium dihydrogen phosphate monohydrate ;di-sodium hydrogen phosphate dihydrate; potassium dihydrogen phosphate;di-potassium hydrogen phosphate; ammonium dihydrogen phosphate;di-ammonium hydrogen phosphate; tri-lithium citrate tetrahydrate;tri-sodium citrate dihydrate; tri-potassium citrate monohydrate;di-ammonium hydrogen citrate; barium chloride; cadmium chloridedihydrate; cobaltous chloride dihydrate; cupric chloride dihydrate;strontium chloride hexahydrate; yttrium chloride hexahydrate; ethyleneglycol; Glycerol anhydrous; 1,6 hexanediol; MPD; polyethylene glycol400; trimethylamine HCl; guanidine HCl; urea; 1,2,3-heptanetriol;benzamidine HCl; dioxane; ethanol; iso-propanol; methanol; sodiumiodide; L-cysteine; EDTA sodium salt; NAD; ATP disodium salt;D(+)-glucose monohydrate; D(+)-sucrose; xylitol; spermidine; sperminetetra-HCl; 6-aminocaproic acid; 1,5-diaminopentane di-HCl;1,6-diaminohexane; 1,8-diaminooctane; glycine; glycyl-glycyl-glycine;hexaminecobalt trichloride; taurine; betaine monohydrate;polyvinylpyrrolidone K15; non-detergent sulfo-betaine 195; non-detergentsulfo-betaine 201; phenol; DMSO; dextran sulfate sodium salt; jeffamineM-600; 2,5 Hexanediol; (+/−)-1,3 butanediol; polypropylene glycol P400;1,4 butanediol; tert-butanol; 1,3 propanediol; acetonitrile; gammabutyrolactone; propanol; ethyl acetate; acetone; dichloromethane;n-butanol; 2,2,2 trifluoroethanol; DTT; TCEP; nonaethylene glycolmonododecyl ether, nonaethylene glycol monolauryl ether,;polyoxyethylene (9) ether; octaethylene glycol monododecyl ether,octaethylene glycol monolauryl ether,; polyoxyethylene (8) lauryl ether;Dodecyl-β-D-maltopyranoside; Lauric acid sucrose ester;Cyclohexyl-pentyl-β-D-maltoside; Nonaethylene glycol octylphenol ether;Cetyltrimethylammonium bromide; N,N-bis(3-D-gluconamidopropyl)-deoxycholamine; Decyl-β-D-maltopyranoside;Lauryldimethylamine oxide; Cyclohexyl-pentyl-β-D-maltoside;n-Dodecylsulfobetaine, 3-(Dodecyldimethylammonio)propane-1-sulfonate;Nonyl-β-D-glucopyranoside; Octyl-β-D-thioglucopyranoside, OSG;N,N-Dimethyldecylamine-β-oxide;Methyl-6-O-(N-heptylcarbamoyl)-a-D-glucopyranoside; Sucrosemonocaproylate; n-Octanoyl-β-D-fructofuranosyl-a-D-glucopyranoside;Heptyl-β-D-thioglucopyranoside; Octyl-β-D-glucopyranoside, OG;Cyclohexyl-propyl-β-D-maltoside;Cyclohexylbutanoyl-N-hydroxyethylglucamide; n-decylsulfobetaine,3-(Decyldimethylammonio)propane-1-sulfonate; Octanoyl-N-methylglucamide,OMEGA; Hexyl-β-D-glucopyranoside; Brij 35; Brij 58; Triton X-114; TritonX-305; Triton X-405; Tween 20; Tween 80; polyoxyethylene(6)decyl ether;polyoxyethylene(9)decyl ether; polyoxyethylene(10)dodecyl ether;polyoxyethylene(8)tridecyl ether; Isopropyl-β-D-thiogalactoside;Decanoyl-N-hydroxyethylglucamide; Pentaethylene glycol monooctyl ether;3-[(3-cholamidopropyl)-dimethylammonio]-1-propane sulfonate;3-[(3-Cholamidopropyl)-dimethylammonio]-2-hydroxy-1-propane sulfonate;Cyclohexylpentanoyl-N-hydroxyethylglucamide;Nonanoyl-N-hydroxyethyglucamide;Cyclohexylpropanol-N-hydroxyethylglucamide;Octanoyl-N-hydroxyethylglucamide;Cyclohexylethanoyl-N-hydroxyethylglucamide; Benzyldimethyldodecylammonium bromide; n-Hexadecyl-β-D-maltopyranoside;n-Tetradecyl-β-D-maltopyranoside; n-Tridecyl-β-D-maltopyranoside;Dodecylpoly(ethyleneglycoether)n;n-Tetradecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate;n-Undecyl-β-D-maltopyranoside; n-Decyl-β-D-thiomaltopyranoside;n-dodecylphosphocholine; a-D-glucopyranoside, β-D-fructofuranosylmonodecanoate, sucrose mono-caprate; 1-s-Nonyl-β-D-thioglucopyranoside;n-Nonyl-β-D-thiomaltoyranoside; N-Dodecyl-N,N-(dimethlammonio)butyrate;n-Nonyl-β-D-maltopyranoside; Cyclohexyl-butyl-β-D-maltoside;n-Octyl-β-D-thiomaltopyranoside; n-Decylphosphocholine;n-Nonylphosphocholine; Nonanoyl-N-methylglucamide;1-s-Heptyl-β-D-thioglucopyranoside; n-Octylphosphocholine;Cyclohexyl-ethyl-β-D-maltoside;n-Octyl-N,N-dimethyl-3-ammonio-1-propanesulfonate;Cyclohexyl-methyl-β-D-maltoside.

Cryosolvents are agents that stabilize a target crystal to flash-coolingin a cryogen such as liquid nitrogen, liquid propane, liquid ethane, orgaseous nitrogen or helium (all at approximately 100–120° K.) such thatcrystal becomes embedded in a vitreous glass rather than ice. Any numberof salts or small molecular weight organic compounds can be used as acryoprotectant, and typical ones include but are not limited to: MPD,PEG-400 (as well as both PEG derivatives and higher molecular-weight PEGcompounds), glycerol, sugars (xylitol, sorbitol, erythritol, sucrose,glucose, etc.), ethylene glycol, alcohols (both short- and long chain,both volatile and nonvolatile), LiOAc, LiCl, LiCHO₂, LiNO₃, Li2SO₄,Mg(OAc)₂, NaCl, NaCHO₂, NaNO₃, etc. Again, materials from whichmicrofluidics devices in accordance with the present invention arefabricated may be compatible with a range of such compounds.

Many of these chemicals can be obtained in predefined screening kitsfrom a variety of vendors, including but not limited to Hampton Researchof Laguna Niguel, Calif., Emerald Biostructures of Bainbridge Island,Wash., and Jena BioScience of Jena, Germany, that allow the researcherto perform both ‘sparse matrix’ and ‘grid’ screening experiments. Sparsematrix screens attempt to randomly sample as much of precipitant,buffer, and additive chemical space as possible with as few conditionsas possible. Grid screens typically consist of systematic variations oftwo or three parameters against one another (e.g., precipitantconcentration vs. pH). Both types of screens have been employed withsuccess in crystallization trials, and the majority of chemicals andchemical combinations used in these screens are compatible with the chipdesign and matrices in accordance with embodiments of the presentinvention.

Moreover, current and future designs of microfluidic devices may enableflexibly combinatorial screening of an array of different chemicalsagainst a particular target or set of targets, a process that isdifficult with either robotic or hand screening. This latter aspect isparticularly important for optimizing initial successes generated byfirst-pass screens.

13. Additional Screening Variables for Crystallization

In addition to chemical variability, a host of other parameters can bevaried during crystallization screening. Such parameters include but arenot limited to: 1) volume of crystallization trial, 2) ratio of targetsolution to crystallization solution, 3) target concentration, 4)cocrystallization of the target with a secondary small or macromolecule,5) hydration, 6) incubation time, 7) temperature, 8) pressure, 9)contact surfaces, 10) modifications to target molecules, and 11)gravity.

Volumes of crystallization trials can be of any conceivable value, fromthe picoliter to milliliter range. Typical values may include but arenot limited to: 0.1, 0.2, 0.25, 0.4, 0.5, 0.75, 1, 2, 4, 5, 10, 15, 20,25, 30, 35, 40, 45, 50, 60, 70, 75, 80, 90, 100, 125, 150, 175, 200,225, 250, 275, 300, 350, 400, 450, 500, 550, 600, 700, 750, 800, 900,1000, 1100, 1200, 1250, 1 300, 1400, 1500, 1600, 1700, 1800, 1900, 2000,2250, 2500, 3000, 4000, 5000, 6000, 7000, 7500, 8000, 9000, and 10000nL. The microfluidics devices previously described can access thesevalues.

In particular, access to the low volume range for crystallization trials(<100 nL) is a distinct advantage of embodiments of the microfluidicschips in accordance with embodiments of the present invention, as suchsmall-volume crystallization chambers can be readily designed andfabricated, minimizing the need the need for large quantities ofprecious target molecules. The low consumption of target material ofembodiments in accordance with the present invention is particularlyuseful in attempting to crystallize scarce biological samples such asmembrane proteins, protein/protein and protein/nucleic acid complexes,and small-molecule drug screening of lead libraries for binding totargets of interest.

The ratios of a target solution to crystallization mix can alsoconstitute an important variable in crystallization screening andoptimization. These rations can be of any conceivable value, but aretypically in the range of 1:100 to 100:1target:crystallization-solution. Typical target:crystallization-solutionor crystallization-solution:target ratios may include but are notlimited to: 1:100, 1:90, 1:80, 1:70, 1:60, 1:50, 1:40, 1:30, 1:25, 1:20,1:15, 1:10, 1:9, 1:8, 1:7.5, 1:7, 1:6, 1:5, 1:4, 1:3, 1:2.5, 1:2, 1:1,2:3, 3:4, 3:5, 4:5, 5:6, 5:7, 5:9, 6:7, 7:8, 8:9, and 9:10. Aspreviously described, microfluidics devices in accordance withembodiments of the present invention can be designed to access multipleratios simultaneously on a single chip.

Target concentration, like crystallization chemical concentration, canlie in a range of values and is an important variable in crystallizationscreening. Typical ranges of concentrations can be anywhere from <0.5mg/ml to >100 mg/ml, with most commonly used values between 5–30 mg/ml.The microfluidics devices in accordance with embodiments of the presentinvention are readily compatible with this range of values.

Cocrystallization generally describes the crystallization of a targetwith a secondary factor that is a natural or nonnatural binding partner.Such secondary factors can be small, on the order of about 10–1000 Da,or may be large macromolecules. Cocrystallization molecules can includebut are not limited to small-molecule enzyme ligands (substrates,products, allosteric effectors, etc.), small-molecule drug leads,single-stranded or double-stranded DNAs or RNAs, complement proteins(such as a partner or target protein or subunit), monoclonal antibodies,and fusion-proteins (e.g, maltose binding proteins, glutathioneS-transferase, protein-G, or other tags that can aid expression,solubility, and target behavior). As many of these compounds are eitherbiological or of a reasonable molecular weight, cocrystallizationmolecules can be routinely included with screens in the microfluidicschips. Indeed, because many of these reagents are expensive and/or oflimited quantity, the small-volumes afforded by the microfluidics chipsin accordance with embodiment of the present invention make them ideallysuited for cocrystallization screening.

Hydration of targets can be an important consideration. In particular,water is by far the dominant solvent for biological targets and samples.The microfluidics devices described in this document are relativelyhydrophobic, and are compatible with water-based solutions.

The length of time for crystallization experiments can range fromminutes or hours to weeks or months. Most experiments on biologicalsystems typically show results within 24 hours to 2 weeks. This regimeof incubation time can be accommodated by the microfluidics devices inaccordance with embodiments of the present invention.

The temperature of a crystallization experiment can have a great impacton success or failure rates. This is particularly true for biologicalsamples, where temperatures of crystallization experiments can rangefrom 0–42° C. Some of the most common crystallization temperatures are:0, 1, 2, 4, 5, 8, 10, 12, 15, 18, 20, 22, 25, 30, 35, 37, and 42.Microfluidics devices in accordance with embodiments of the presentinvention can be stored at the temperatures listed, or alternatively maybe placed into thermal contact with small temperature control structuressuch as resistive heaters or Peltier cooling structures.

In addition, the small footprint and rapid setup time of embodiments inaccordance with the present invention allow faster equilibration todesired target temperatures and storage in smaller incubators at a rangeof temperatures. Moreover, as the microfluidics systems in accordancewith embodiments of the present invention do not place thecrystallization experiment in contact with the vapor phase, condensationof water from the vapor phase into the drop as temperatures change, aproblem associated with conventional macroscopic vapor-diffusiontechniques, is avoided. This feature represents an advance over manyconventional manual or robotic systems, where either the system must bemaintained at the desired temperature, or the experiment must remain atroom temperature for a period before being transferred to a newtemperature.

Variation in pressure is an as yet understudied crystallizationparameter, in part because conventional vapor-diffusion and microbatchprotocols do not readily allow for screening at anything typically otherthan atmospheric pressure. The rigidity of the PDMS matrix enablesexperiments to probe the effects of pressure on target crystallizationon-chip.

The surface on which the crystallization ‘drop’ sits can affectexperimental success and crystal quality. Examples of solid supportcontact surfaces used in vapor diffusion and microbatch protocolsinclude either polystyrene or silanized glass. Both types of supportscan show different propensities to promote or inhibit crystal growth,depending on the target. In addition, the crystallization ‘drop’ is incontact with either air or some type of poly-carbon oil, depending onwhether the experiment is a vapor-diffusion or microbatch setup,respectively. Air contact has the disadvantage in that free oxygenreacts readily with biological targets, which can lead to proteindenaturation and inhibit or degrade crystallization success. Oil allowstrace hydrocarbons to leach into the crystallization experiment, and cansimilarly inhibit or degrade crystallization success.

Microfluidics device designs in accordance with embodiments of thepresent invention may overcome these limitations by providing anonreactive, biocompatible environment that completely surrounds thecrystallization reaction. Moreover, the composition of thecrystallization chambers in the microfluidics chips can conceivably bevaried to provide new surfaces for contacting the crystallizationreaction; this would allow for routine screening of different surfacesand surface properties to promote crystallization.

Crystallization targets, particularly those of biological origin, mayoften be modified to enable crystallization. Such modifications includebut are not limited to truncations, limited proteolytic digests,site-directed mutants, inhibited or activated states, chemicalmodification or derivatization, etc. Target modifications can be timeconsuming and costly; modified targets require the same thoroughscreening as do unmodified targets. Microfluidics devices of the presentinvention work with such modified targets as readily as with theoriginal target, and provide the same benefits.

The effect of gravity as a parameter for crystallization is yet anotherunderstudied crystallization parameter, because of the difficulty ofvarying such a physical property. Nonetheless, crystallizationexperiments of biological samples in zero gravity environments haveresulted in the growth of crystals of superior quality than thoseobtained on Earth under the influence of gravity.

The absence of gravity presents problems for traditional vapor-diffusionand microbatch setups, because all fluids must be held in place bysurface tension. The need to often set up such experiments by hand alsoposes difficulties because of the expense of maintaining personnel inspace. Microfluidics devices in accordance with embodiments of thepresent invention, however, would enable further exploration ofmicrogravity as a crystallization condition. A compact, automatedmetering and crystal growth system would allow for: 1) launching ofsatellite factory containing target molecules in a cooled, but liquidstate, 2) distribution of targets and growth of crystals, 3) harvestingand cryofreezing of resultant crystals, and 4) return of cryo-storedcrystals to land-based stations for analysis.

14. In situ Crystallization Screening

The ability to observe the growth of crystals with a microscope is astep in deciding upon success or failure of crystallization trials.Conventional crystallization protocols may use transparent materialssuch as polystyrene or silanized glass to allow for visualization. Thetransparency of the PDMS matrix of embodiments in accordance with thepresent invention is particularly suited to the two primary methods bywhich crystallization trials are traditionally scored: 1) directobservation in the visible light regime by optical microscopes and 2)birefringence of polarized light.

Birefringence may be difficult to judge in conventional experiments asmany plastics are themselves birefringent, interfering with sampleassessment. However, the microfluidics devices described herein can bemade without such optical interference properties, allowing for thedesign of an automated scanning system that routinely allows directvisualization with both polarizing and non-polarizing features.

In addition, robotic and, in particular, manually-set crystallizationexperiments can vary the placement of a crystallization drop on asurface by tens to hundreds of microns. This variability presents aproblem for automated scanning systems, as it is difficult to program inthe need for such flexible positioning without stable fiducials.However, the fixed placement of crystallization chambers in themicrofluidics chips of embodiments of the present invention overcomessuch problems, as every well can be positioned in a particular locationwith submicron accuracy. Moreover, such a system is readily scalable forthe design of differently sized and positioned crystallization chambers,as masks and other templates used to design microfluidics devices inaccordance with embodiments of the present invention can be simplydigitized and ported into scanning software for visualization.

Once crystals are obtained by visual inspection, it may be possible toscreen for diffraction directly through the chip itself. For example, acrystallization chamber within a chip could be outfitted withtransparent ‘windows’ comprising glass, quartz, or thinned portions ofthe the elastomer material itself, on opposite walls of the chamber.Crystals could then be exposed directly to x-rays through the chip toassay for diffraction capabilities, eliminating the need to remove, andthereby possibly damage, the crystalline sample. Such an approach couldbe used to screen successes from initial crystallization trials todetermine the best starting candidate conditions for follow-up study.Similarly, crystals grown under a particular set of conditions could be‘re-equilibrated’ with new solutions (e.g., cryo-stabilizing agents,small-molecule drug leads or ligands, etc.), and the stability of thecrystals to such environment changes monitored directly by x-raydiffraction.

15. Utilizing Microfluidic Devices for Purification/Crystallization

Crystallization of target biological samples such as proteins isactually the culmination of a large number of prior complex anddifficult steps, including but not limited to protein expression,purification, derivitization, and labeling. Such steps prior tocrystallization comprise shuttling liquids from a chamber with one setof solution properties to another area with a different set ofproperties. Mircofluidics technology is suited to perform such tasks,allowing for the combination of all necessary steps within the confinesof a single chip.

Examples of microfluidic handling structures enabling performance ofpre-crystallization steps have been described under section I above. Forexample, a microfluidics chip could act as a regulated bioreactor,allowing nutrients to flow into growing cells contained in cell penstructure while removing wastes and inducing recombinantly-modifiedorganisms to produce target molecules (e.g., proteins) at a desiredstage of cell growth. Following induction, these cells could be shuntedfrom the cell pen to a different region of the chip for lysis byenzymatic or mechanical means. Solubilized target molecules could thenbe separated from cellular debris by molecular filtration unitsincorporated directly onto a chip.

The crude mixture of target molecules and contaminating cellularproteins and nucleic acids could then be funneled through porousmatrices of differing chemical properties (e.g., cation-exchange,anion-exchange, affinity, size-exclusion) to achieve separation. If atarget molecule were tagged with a fusion protein of a particular typeto promote solubility, it could be affinity purified, briefly treatedwith a similarly-tagged, site-specific protease to separate the fusionproduct, and then repassaged though the affinity matrix as a clean-upstep.

Once pure, the target could be mixed with different stabilizing agents,assayed for activity, and then transported to crystallization stagingareas. Localized heating (such as an electrode) and refrigeration (suchas a Peltier cooler) units stationed at various points on a chip or achip holder would allow for differential temperature regulation at allstages throughout the processing and crystallization. Thus, theproduction, purification, and crystallization of proteins may beaccomplished on an embodiment of a single microfluidics device inaccordance with the present invention.

16. Applications Other Than Crystallization Screening

Thus far, the instant application has focused upon the ability ofmicrofluidics devices in accordance with embodiments of the presentinvention to meter small volumes of material in the context ofperforming crystallization of target material. However, embodiments ofmicrofluidic structures in accordance with the present invention may beemployed for other applications. Examples of such applications aresummarized below. A more complete description of possible applicationsmay be found in PCT application PCT/US01/44869, filed Nov. 16, 2001 andentitled “Cell Assays and High Throughput Screening”, herebyincorporated by reference for all purposes. Examples of microfluidicstructures suitable for performing such applications include thosedescribed herein, as well as others described in U.S. nonprovisionalpatent application No. 60/334473, “Nucleic Acid Amplification UtilizingMicrofluidic Devices”, filed Apr. 5, 2002, hereby incorporated byreference for all purposes.

A wide variety of binding assays can be conducted utilizing themicrofluidic devices disclosed herein. Interactions between essentiallyany ligand and antiligand can be detected. Examples of ligand/antiligandbinding interactions that can be investigated include, but are notlimited to, enzyme/ligand interactions (e.g., substrates, cofactors,inhibitors); receptor/ligand; antigen/antibody; protein/protein(homophilic/heterophilic interactions); protein/nucleic; DNA/DNA; andDNA/RNA. Thus, the assays can be used to identify agonists andantagonists to receptors of interest, to identify ligands able to bindreceptors and trigger an intracellular signal cascade, and to identifycomplementary nucleic acids, for example. Assays can be conducted indirect binding formats in which a ligand and putative antiligand arecontacted with one another or in competitive binding formats well knownto those of ordinary skill in the art.

Heterogenous binding assays involve a step in which complexes areseparated from unreacted agents so that labeled complexes can bedistinguished from uncomplexed labeled reactants. Often this is achievedby attaching either the ligand or antiligand to a support. After ligandsand antiligands have been brought into contact, uncomplexed reactantsare washed away and the remaining complexes subsequently detected.

The binding assays conducted with the microfluidic devices providedherein can also be conducted in homogeneous formats. In the homogeneousformats, ligands and antiligands are contacted with one another insolution and binding complexes detected without having to removeuncomplexed ligands and antiligands. Two approaches frequently utilizedto conduct homogenous assays are fluorescence polarization (FP) and FRETassays.

The microfluidic devices can also be utilized in a competitive formatsto identify agents that inhibit the interaction between known bindingpartners. Such methods generally involve preparing a reaction mixturecontaining the binding partners under conditions and for a timesufficient to allow the binding partners to interact and form a complex.In order to test a compound for inhibitory activity, the reactionmixture is prepared in the presence (test reaction mixture) and absence(control reaction mixture) of the test compound. Formation of complexesbetween binding partners is then detected, typically by detecting alabel borne by one or both of the binding partners. The formation ofmore complexes in the control reaction then in the test reaction mixtureat a level that constitutes a statistically significant differenceindicates that the test compound interferes with the interaction betweenthe binding partners.

Immunological assays are one general category of assays that can beperformed with the microfluidic devices in accordance with embodimentsof the present invention. Certain assays are conducted to screen apopulation of antibodies for those that can specifically bind to aparticular antigen of interest. In such assays, a test antibody orpopulation of antibodies is contacted with the antigen. Typically, theantigen is attached to a solid support. Examples of immunological assaysinclude enzyme linked immunosorbent assays (ELISA) and competitiveassays as are known in the art.

Utilizing the microfluidic devices provided herein, a variety ofenzymatic assays can be performed. Such enzymatic assays generallyinvolve introducing an assay mixture containing the necessary componentsto conduct an assay into the various branch flow channels. The assaymixtures typically contain the substrate(s) for the enzyme, necessarycofactors (e.g., metal ions, NADH, NAPDH), and buffer, for example. If acoupled assay is to be performed, the assay solution will also generallycontain the enzyme, substrate(s) and cofactors necessary for theenzymatic couple.

Microfluidic devices in accordance with embodiments of the presentinvention can be arranged to include a material that selectively bindsto an enzymatic product that is produced. In some instances, thematerial has specific binding affinity for the reaction product itself.Somewhat more complicated systems can be developed for enzymes thatcatalyze transfer reactions. Certain assays of this type, for example,involve incubating an enzyme that catalyzes the transfer of a detectablemoiety from a donor substrate to an acceptor substrate that bears anaffinity label to produce a product bearing both the detectable moietyand the affinity label. This product can be captured by material thatincludes a complementary agent that specifically binds to the affinitylabel. This material typically is located in a detection region suchthat captured product can be readily detected. In certain assays, thematerial is coated to the interior channel walls of the detectionsection; alternatively, the material can be a support located in thedetection region that is coated with the agent.

Certain assays utilizing the present devices are conducted with vesiclesrather than cells. Once example of such an assay is a G-protein coupledreceptor assay utilizing fluorescent correlation spectroscopy (FCS).Membrane vesicles constructed from cells that over-express the receptorof interest are introduced into a main flow channel. Vesicles can eitherbe premixed with inhibitor and introduced via branch flow channels orvia one of the main flow channels prior to being mixed with afluorescent natural ligand which is also introduced by a main flowchannel. Components are allowed to incubate for the desired time andfluorescent signals may be analyzed directly in the flow chamber usingan FCS reader such as the Evotec/Zeiss Confocor (a single or dual photoncounting device).

FRET assays can also be utilized to conduct a number of ligand-receptorinteractions using the devices disclosed herein. For example, a FRETpeptide reporter can be constructed by introducing a linker sequence(corresponding to an inducible domain of a protein such as aphosphorylation site) into a vector encoding for a fluorescent proteincomposed of blue- and red-shifted GFP variants. The vector can be abacterial (for biochemical studies) or a mammalian expression vector(for in vivo studies).

Assays of nuclear receptors can also be performed with the presentmicrofluidic devices. For example, FRET-based assays forco-activator/nuclear receptor interaction can be performed. As aspecific example, such assays can be conducted to detect FRETinteractions between: (a) a ligand binding domain of a receptor taggedwith CFP (cyan fluorescent protein, a GFP derivative), and (b) areceptor binding protein (a coactivator) tagged with the Yellowfluorescent protein (YFP).

Fluorescence polarization (FP) can be utilized to develop highthroughput screening (HTS) assays for nuclear receptor-liganddisplacement and kinase inhibition. Because FP is a solution-based,homogeneous technique, there is no requirement for immobilization orseparation of reaction components. In general, the methods involve usingcompetition between a fluorescently labeled ligand for the receptor andrelated test compounds.

A number of different cell reporter assays can be conducted with theprovided microfluidic devices. One common type of reporter assay thatcan be conducted include those designed to identify agents that can bindto a cellular receptor and trigger the activation of an intracellularsignal or signal cascade that activates transcription of a reporterconstruct. Such assays are useful for identifying compounds that canactivate expression of a gene of interest. Two-hybrid assays, discussedbelow, are another major group of cell reporter assays that can beperformed with the devices. The two-hybrid assays are useful forinvestigating binding interactions between proteins.

Often cell reporter assays are utilized to screen libraries ofcompounds. In general such methods involve introducing the cells intothe main flow channel so that cells are retained in the chambers locatedat the intersection between the main flow channel and branch channels.Different test agents (e.g., from a library) can then be introduced intothe different branch channels where they become mixed with the cells inthe chambers. Alternatively, cells can be introduced via the main flowchannel and then transferred into the branch channel, where the cellsare stored in the holding areas. Meanwhile, different test compounds areintroduced into the different branch flow channels, usually to at leastpartially fill the chambers located at the intersection of the main andbranch flow channels. The cells retained in the holding area can bereleased by opening the appropriate valves and the cells transferred tothe chambers for interaction with the different test compounds. Once thecells and test compounds have been mixed, the resulting solution isreturned to the holding space or transported to the detection sectionfor detection of reporter expression. The cells and test agents canoptionally be further mixed and incubated using mixers of the design setforth above.

Cells utilized in screening compounds to identify those able to triggergene expression typically express a receptor of interest and harbor aheterologous reporter construct. The receptor is one which activatestranscription of a gene upon binding of a ligand to the receptor. Thereporter construct is usually a vector that includes a transcriptionalcontrol element and a reporter gene operably linked thereto. Thetranscriptional control element is a genetic element that is responsiveto an intracellular signal (e.g., a transcription factor) generated uponbinding of a ligand to the receptor under investigation. The reportergene encodes a detectable transcriptional or translational product.Often the reporter (e.g., an enzyme) can generate an optical signal thatcan be detected by a detector associated with a microfluidic device.

A wide variety of receptor types can be screened. The receptors oftenare cell-surface receptors, but intracellular receptors can also beinvestigated provided the test compounds being screened are able toenter into the cell. Examples of receptors that can be investigatedinclude, but are not limited to, ion channels (e.g., calcium, sodium,potassium channels), voltage-gated ion channels, ligand-gated ionchannels (e.g., acetyl choline receptors, and GABA (gamma-aminobutyricacid) receptors), growth factor receptors, muscarinic receptors,glutamate receptors, adrenergic receptors, dopamine receptors.

Another general category of cell assays that can be performed is the twohybrid assays. In general, the two-hybrid assays exploit the fact thatmany eukaryotic transcription factors include a distinct DNA—bindingdomain and a distinct transcriptional activation domain to detectinteractions between two different hybrid or fusion-proteins. Thus, thecells utilized in two-hybrid assays include the construct(s) that encodefor the two fusion proteins. These two domains are fused to separatebinding proteins potentially capable of interacting with one anotherunder certain conditions. The cells utilized in conducting two-hybridassays contain a reporter gene whose expression depends upon either aninteraction, or lack of interaction, between the two fusion proteins.

In addition to the assays just described, a variety of methods to assayfor cell membrane potential can be conducted with the microfluidicdevices disclosed herein. In general, methods for monitoring membranepotential and ion channel activity can be measured using two alternatemethods. One general approach is to use fluorescent ion shelters tomeasure bulk changes in ion concentrations inside cells. The secondgeneral approach is to use of FRET dyes sensitive to membrane potential.

The microfluidic devices disclosed herein can be utilized to conduct avariety of different assays to monitor cell proliferation. Such assayscan be utilized in a variety of different studies. For example, the cellproliferation assays can be utilized in toxicological analyses, forexample. Cell proliferation assays also have value in screeningcompounds for the treatment of various cell proliferation disordersincluding tumors.

The microfluidic devices disclosed herein can be utilized to perform avariety of different assays designed to identify toxic conditions,screen agents for potential toxicity, investigate cellular responses totoxic insults and assay for cell death. A variety of differentparameters can be monitored to assess toxicity. Examples of suchparameters include, but are not limited to, cell proliferation,monitoring activation of cellular pathways for toxicological responsesby gene or protein expression analysis, DNA fragmentation; changes inthe composition of cellular membranes, membrane permeability, activationof components of death-receptors or downstream signaling pathways (e.g.,caspases), generic stress responses, NF-kappaB activation and responsesto mitogens. Related assays are used to assay for apoptosis (aprogrammed process of cell death) and necrosis.

By contacting various microbial cells with different test compounds, onecan also utilize the devices provided herein to conduct antimicrobialassays, thereby identifying potential antibacterial compounds. The term“microbe” as used herein refers to any microscopic and/or unicellularfungus, any bacteria or any protozoan. Some antimicrobial assays involveretaining a cell in a cell cage and contacting it with at least onepotential antimicrobial compound. The effect of the compound can bedetected as any detectable change in the health and/or metabolism of thecell. Examples of such changes, include but are not limited to,alteration in growth, cell proliferation, cell differentiation, geneexpression, cell division and the like.

Certain of the microfluidic devices provided herein can be utilized toconduct mini-sequencing reactions or primer extension reactions toidentify the nucleotide present at a polymorphic site in a targetnucleic acid. In general, in these methods a primer complementary to asegment of a target nucleic acid is extended if the reaction isconducted in the presence of a nucleotide that is complementary to thenucleotide at the polymorphic site. Often such methods are single basepair extension (SBPE) reactions. Such method typically involvehybridizing a primer to a complementary target nucleic acid such thatthe 3′ end of the primer is immediately adjacent the polymorphic site,or is a few bases upstream of the polymorphic site. The extensionreaction is conducted in the presence of one or more labelednon-extendible nucleotides (e.g., dideoxynucleotides) and a polymerase.Incorporation of a non-extendible nucleotide onto the 3′ end of theprimer prevents further extension of the primer by the polymerase oncethe non-extendible nucleotide is incorporated onto the 3′ end of theprimer.

Related to the methods just described, the present devices can also beutilized to amplify and subsequently identify target nucleic acids inmultiple samples using amplification techniques that are wellestablished in the art. In general such methods involve contacting asample potentially containing a target nucleic acid with forward andreverse primers that specifically hybridize to the target nucleic acid.The reaction includes all four dNTPs and polymerase to extend the primersequences.

An embodiment of a method of fabricating a microfluidic device inaccordance with the present invention comprises etching a top surface ofa glass substrate to produce a plurality of wells, molding an elastomerblock such that a bottom surface bears a patterned recess, placing abottom surface of the molded elastomer block into contact with the topsurface of the glass substrate, such that the patterned recess isaligned with the wells to form a flow channel between the wells.

An embodiment of a method for forming crystals of a target materialcomprises priming a first chamber of an elastomeric microfluidic devicewith a first predetermined volume of a target material solution. Asecond chamber of an elastomer microfluidic device is primed with asecond predetermined volume of a crystallizing agent. The first chamberis placed into fluidic contact with the second chamber to allowdiffusion between the target material and the crystallizing agent, suchthat an environment of the target material is changed to cause formationof crystal.

While the present invention has been described herein with reference toparticular embodiments thereof, a latitude of modification, variouschanges and substitutions are intended in the foregoing disclosure, andit will be appreciated that in some instances some features of theinvention will be employed without a corresponding use of other featureswithout departing from the scope of the invention as set forth.Therefore, many modifications may be made to adapt a particularsituation or material to the teachings of the invention withoutdeparting from the essential scope and spirit of the present invention.It is intended that the invention not be limited to the particularembodiment disclosed as the best mode contemplated for carrying out thisinvention, but that the invention will include all embodiments andequivalents falling within the scope of the claims.

1. A method for promoting interaction between two solutions, the methodcomprising: defining a first microfluidic chamber; priming the firstmicrofluidic chamber with a first solution; defining a secondmicrofluidic chamber; priming the second microfluidic chamber with asecond solution; placing the first microfluidic chamber into fluidcommunication with the second microfluidic chamber to define amicrofluidic free interface between the first solution and the secondsolution; and permitting free-interface diffusion to occur between thefirst solution and the second solution such that the first solutioninteracts with the second solution.
 2. The method of claim 1 wherein:the first solution comprises a crystallization target material solution;the second solution comprises a crystallizing agent solution; and theinteraction comprises alteration of a solvent environment of the targetmaterial to promote crystallization of the target material.
 3. Themethod of claim 1 wherein: the first solution comprises a samplesolution; the second solution comprises a reagent solution; and theinteraction comprises a chemical reaction between the sample and thereagent.
 4. The method of claim 1 wherein defining the first chamber andthe second chamber comprises placing an elastomer block bearing a firstpatterned recess on a lower surface into contact with a substantiallyplanar substrate.
 5. The method of claim 1 wherein defining the firstchamber and the second chamber comprises placing a substantially planarlower surface of an elastomer block into contact with a substratebearing a first patterned recess.
 6. The method of claim 1 whereindefining the first chamber and the second chamber comprises placing anelastomer block bearing a first patterned recess on a lower surface intocontact with a substrate bearing a second patterned recess, the firstpatterned recess aligned with the second patterned recess.
 7. The methodof claim 1 wherein: the elastomer material is permeable to a gas;priming the first microfluidic chamber comprises introducing the firstsolution solution under pressure into the first microfluidic chamber todisplace into the surrounding elastomer the gas formerly occupying thefirst chamber, thereby enabling substantially complete filling of thefirst chamber; and priming the second microfluidic chamber comprisesintroducing the second solution under pressure into the secondmicrofluidic chamber to displace into the surrounding elastomer the gasformerly occupying the second chamber, thereby enabling substantiallycomplete filling of the second chamber.
 8. The method of claim 1 whereinthe first chamber and the second chamber are placed into fluidcommunication through a microfluidic flow channel defined between theelastomer block and the substrate.
 9. The method of claim 8 wherein thechambers are placed into fluidic communication through actuation of avalve present in the microfluidic flow channel.
 10. The method of claim9 wherein the chambers are placed into fluidic communication throughdeactuation of a normally-open valve structure, such that application ofa reduced pressure to a control recess crossing over the flow channel inthe elastomer block causes an elastomer membrane defined between thecontrol recess and the flow channel to relax out of the flow channel todefine the microfluidic free interface.
 11. The method of claim 9wherein the chambers are placed into fluidic communication throughactuation of a normally-closed valve structure, such that application ofa reduced pressure to a control recess crossing over the flow channel inthe elastomer block causes an elastomer membrane defined between thecontrol recess and the flow channel to be deflected out of the flowchannel into the control channel to define the microfluidic freeinterface.
 12. A method of capturing a concentration gradient betweentwo fluids, the method comprising: providing a first fluid on a firstside of an elastomer membrane present within a microfluidic flowchannel; providing a second fluid on a second side of the elastomermembrane; displacing the elastomer membrane from the microfluidic flowchannel to define a microfluidic free interface between the first fluidand the second fluid; allowing the first fluid and the second fluid todiffuse across the microfluidic free interface; and actuating a group ofelastomer valves positioned along the flow channel at increasingdistances from the microfluidic free interface to define a succession ofchambers whose relative concentration of the first fluid and the secondfluid reflects a time of diffusion.
 13. The method of claim 12 whereinthe first fluid is a crystallizing agent and the second fluid is acrystallization target solution, such that a plurality of crystallizingconditions is created in the succession of chambers.
 14. A method forforming crystals of a target material comprising: priming a firstchamber of an elastomeric microfluidic device with a first predeterminedvolume of a target material solution; priming a second chamber of anelastomer micro fluidic device with a second predetermined volume of acrystallizing agent solution; and placing the first chamber into fluidiccontact with the second chamber to allow free-interface diffusionbetween the target material and the crystallizing agent solution, suchthat an environment of the target material is changed to cause formationof crystal.
 15. The method of claim 14 wherein the first and secondvolumes are predetermined by dimensions of the first chamber and thesecond chamber.
 16. The method of claim 14 further comprising harvestingthe crystal from the microfluidic device to use as a seed crystal foradditional crystallization screening.
 17. The method of claim 14 furthercomprising irradiating the crystal within the microfluidic device withx-ray radiation to determine a three dimensional structure of thecrystal.
 18. The method of claim 14 further comprising delivering acryogen to the microfluidic device prior to irradiating the crystal. 19.The method of claim 14 wherein: a concentration and volume of materialwithin the first and second chambers represents one of a plurality ofcrystallization conditions within the microfluidic device; and themethod further comprises performing a second crystallization screeningon a second microfluidic chip under a set of conditions related to theone of the plurality of crystallization conditions.